A Phase I Double Blind, Placebo-Controlled, Randomized Study of the Safety and Immunogenicity of Electroporated HIV DNA with or without Interleukin 12 in Prime-Boost Combinations with an Ad35 HIV Vaccine in Healthy HIV-Seronegative African Adults

Background Strategies to enhance the immunogenicity of DNA vaccines in humans include i) co-administration of molecular adjuvants, ii) intramuscular administration followed by in vivo electroporation (IM/EP) and/or iii) boosting with a different vaccine. Combining these strategies provided protection of macaques challenged with SIV; this clinical trial was designed to mimic the vaccine regimen in the SIV study. Methods Seventy five healthy, HIV-seronegative adults were enrolled into a phase 1, randomized, double-blind, placebo-controlled trial. Multi-antigenic HIV (HIVMAG) plasmid DNA (pDNA) vaccine alone or co-administered with pDNA encoding human Interleukin 12 (IL-12) (GENEVAX IL-12) given by IM/EP using the TriGrid Delivery System was tested in different prime-boost regimens with recombinant Ad35 HIV vaccine given IM. Results All local reactions but one were mild or moderate. Systemic reactions and unsolicited adverse events including laboratory abnormalities did not differ between vaccine and placebo recipients. No serious adverse events (SAEs) were reported. T cell and antibody response rates after HIVMAG (x3) prime—Ad35 (x1) boost were independent of IL-12, while the magnitude of interferon gamma (IFN-γ) ELISPOT responses was highest after HIVMAG (x3) without IL-12. The quality and phenotype of T cell responses shown by intracellular cytokine staining (ICS) were similar between groups. Inhibition of HIV replication by autologous T cells was demonstrated after HIVMAG (x3) prime and was boosted after Ad35. HIV specific antibodies were detected only after Ad35 boost, although there was a priming effect with 3 doses of HIVMAG with or without IL-12. No anti-IL-12 antibodies were detected. Conclusion The vaccines were safe, well tolerated and moderately immunogenic. Repeated administration IM/EP was well accepted. An adjuvant effect of co-administered plasmid IL-12 was not detected. Trial Registration ClinicalTrials.gov NCT01496989

Healthy male or female adults, 18 to 50 years of age, who do not report high-risk behaviour for HIV infection, who are available for the duration of the trial, who are willing to undergo HIV testing, use an effective method of contraception, and who, in the opinion of the principal investigator or designee, understand the study and who provide written informed consent.
Principal exclusion criteria include confirmed HIV infection, pregnancy and lactation, significant acute or chronic disease, clinically significant laboratory abnormalities, recent vaccination or receipt of a blood product, previous receipt of an HIV vaccine, and previous severe local or systemic reactions to vaccination or history of severe allergic reactions.

NUMBER OF VOLUNTEERS:
Approximately 75 volunteers (60 vaccine/15 placebo recipients) will be included in the study. An over-enrolment of up to 10% (up to 83 volunteers total) will be permitted in the study to facilitate rapid enrolment.

FORMULA-TIONS, VOLUMES and ROUTES of INJECTIONS,
HIV-MAG vaccine consisting of two DNA plasmids (ProfectusVax HIV-1 gag/pol DNA plasmid and ProfectusVax HIV-1 nef/tat/vif, env DNA plasmid) are formulated in 30mM citrate buffer (pH 6.5) containing 150 mM NaCl, 0.01% EDTA, and 0.25% bupivacaine HCl in separate vials. The two plasmids are mixed immediately prior to administration.
All administrations of HIV-MAG with GENEVAX® IL-12 (or placebo) consist of 2 IM injections, one into each medial deltoid, by in vivo electroporation (EP) using the Ichor Medical Systems TriGrid™ Delivery System (TDS-IM).
Ad35-GRIN/ENV containing two Ad35 constructs (Ad35-GRIN and Ad35-ENV) are co-formulated in Tris 10 mM pH 8.5, Sucrose 342.3 g/L, 1mM MgCl 2 , Tween80 54 mg/L and 150mM NaCl in the same vial. All administrations of Ad35-GRIN/ENV (or placebo) consist of one IM injection in the deltoid muscle. At the end of the study, a full analysis will be prepared according to a pre-specified statistical analysis plan. In addition, there may also be interim reviews of blinded data. All clinical and routine laboratory data will be included in the safety analysis. Immunogenicity analysis will be performed according to a predefined analysis plan for all volunteers who received vaccine or placebo.

SIGNATURE PAGE
The signatures below constitute the approval of this protocol and the appendices and provide the necessary assurances that this study will be conducted in compliance with the protocol, Good Clinical Practice (GCP) and the applicable regulatory requirement(s).  4 . These results suggest that more potent vaccine regimens will be required to generate HIV-1 immune responses providing more significant protection 5 .
Experimental and natural history studies suggest that both HIV-specific neutralizing/functional antibodies and long-lasting effector and central memory HIV-specific CD8+ and CD4+ T-cell responses are needed in both systemic and mucosal compartments to effectively control HIV infection 6 7 8 9 10 11 12 13 .
Neutralizing antibodies against circulating isolates are induced principally by the envelope glycoprotein (Env) of HIV and could potentially confer sterilizing immunity against HIV, as suggested by non-human primate SHIV challenge studies 14 15 16 . However, attempts to design appropriate immunogens have failed. This failure has been a major drawback for env-based HIV vaccines, since current immunogens afford only very narrow protection against HIV strains that are closely related to the vaccine antigen 17 18 . The search for an immunogen able to induce broad cross-protective and long-lasting neutralizing/functional antibodies remains difficult and critical 19 20 .
Evidence that CD8+ cytotoxic T lymphocytes (CTL) can control HIV replication in the absence of antibodies has been demonstrated in HIV-infected subjects with a reduction in viremia in acute infection temporally associated with HIV-1-specific CTL 21 22 23 . The role of CTL was further IAVI Protocol B004, Version 1.0 Page 15 of 79 30Jun11 suggested in the SIV macaque model 24 25 26 27 28 .
Issues of quantity, quality and location impact the ability of CD8+ T cells to mediate protection from infection 29 . To protect from viruses that remain within the cells of the host and cause persistent infection, such as HIV, the immune system has evolved CD4+ and CD8+ T cells. Both T cell subtypes are recruited in tissues by dendritic cells sensitized through the Toll-like receptors to the presence of pathogens. CD4+ T cells provide help to naive CD8+ T cells so they can proliferate and acquire the ability to recognize foreign antigens presented on the surface of the infected cells and kill them. Naive CD8+ and CD4+ T cells become memory T cells once they have encountered the antigen. There are two main subtypes, effector and central memory CD8+ T cells, that can be differentiated by their surface expression of receptors, cytokine production and ability to proliferate 30 .
Preservation and/or restoration of intestinal CD4+ memory T cells seems to be associated with protection from challenge and control of viremia in the non-human primate SIV challenge model 31 . These results suggest that protection against pathogenic lentiviral infection or disease progression may correlate with preservation of mucosal CD4+ T cells. In animals, accumulating evidence suggests that HIV-specific CD4+ T cells are equally crucial for the induction of a protective immune response against HIV 32 . Furthermore, the analysis of immune responses in HIV-infected individuals suggests a crucial role for CD4+ T cells 33 . CD4+ T cells provide essential help to CD8+ effector T cells in long-term non-progressors 34 35 36 . In contrast, the absence of HIV-specific CD4+ T cells in chronically infected individuals seems to be related to an impairment of CD8+ T cell maturation 37 . Altogether, these data suggest that an effective HIV vaccine that controls viral replication should induce strong HIV-specific CD4+ and CD8+ T cell responses.
A heterologous prime-boost vaccination regimen involves priming the immune system to a target antigen delivered by one vaccine and then selectively boosting the immune response by repeat administration of the antigen by a second and distinct vaccine 38 . The synergistic enhancement of immunity to the target antigen is reflected in an increased number of antigenspecific T cells, selective enrichment of high avidity T cells and increased efficacy against pathogenic challenge 39 . Heterologous HIV immunogens derived from different clades for sequential priming and boosting predominantly stimulated T-cell immunity against conserved epitopes 40 , whereas a single vaccine derived from one clade or the mixture of multiple vaccines from different clades primarily raised T-cells against less conserved or non-conserved epitopes 41 42 .
This study proposes to test a prime-boost regimen to elicit both HIV-specific CD4+ T cells and CD8+ T cells with a recombinant Clade B multiantigen HIV pDNA (HIV-MAG) vaccine coadministered with IL-12 pDNA (GENEVAX® IL-12) given intramuscularly by electroporation, followed by a recombinant adenovirus serotype 35-based vector (Ad35-GRIN/ENV, Clade A fusion gene gag, reverse transcriptase, integrase and nef and gp140 gene). In this study, the immune response induced when Ad35GRIN/ENV given as a prime followed by HIV pDNA (HIV-MAG) vaccine co-administered with IL-12 pDNA given intramuscularly by electroporation will also be evaluated.
Ad35-GRIN/ENV was selected as both prime and boost vaccine, as the ongoing Phase 1 clinical study of Ad35-GRIN/ENV (IAVI B001) suggests that the vaccine is generally welltolerated at the dosage proposed in this study, and that the vaccine is immunogenic after one dose 43 .

Study Rationale
We propose to conduct a Phase 1 randomized, placebo-controlled, double-blind clinical trial in HIV-uninfected healthy adult volunteers at low-risk for HIV infection to evaluate the safety, tolerability and immunogenicity of a fixed dose of the HIV-MAG pDNA encoding gag-pol, neftat-vif and env genes, co-administered with two dosage levels of plasmid human IL-12 (IL-12 pDNA) delivered IM/EP, followed by recombinant Ad35-GRIN/ENV HIV vaccine delivered IM. The hypotheses tested for this prime-boost regimen are: • The proposed prime-boost vaccine regimens will be safe.
• GENEVAX ® IL-12 co-administration with HIV-MAG will increase HIV vaccine specific responses. • The Ad35 GRIN /ENV vaccine will boost HIV-specific CD4+ and CD8+ T-cells in a majority of vaccine recipients. • HIV-MAG co-administered with GENEVAX ® IL-12 will boost HIV-specific immune responses induced by the Ad35-GRIN/ENV vaccine.
Plasmid DNA vaccines have been tested in a variety of investigational clinical settings, and given alone, they have been weakly immunogenic in human trials. Various strategies have been used to improve the immunogenicity of DNA vaccines: i) Electroporation (EP) has been shown to be an efficient means to introduce DNA into cells 44  Taken together, the efficacy data from this NHP SIV challenge study and the immunogenicity data from the HVTN 080 Phase 1 clinical study provide a strong rationale to initiate clinical testing of HIV-MAG + GENEVAX® IL-12 prime given IM/EP, followed by an Ad35-GRIN/ENV boost.

Experience with HIV-MAG
HIV-MAG, the HIV-1 multiantigen pDNA vaccine 51 , alone or co-administered with GENEVAX® IL-12, given IM by electroporation or standard needle injection is currently being tested in HIVinfected individuals in the US, but no safety data are available yet (ACTG 5281).
Pre-clinical data in rabbits indicate that the 2 HIV-MAG pDNA constructs, expressing a Gag/Pol fusion protein, a Nef/Tat/Vif fusion protein and Env protein, co-administered with human IL-12 pDNA given either IM or IM/EP are well-tolerated and do not cause any adverse effects. No significant differences in biodistribution or persistence were observed when vaccine pDNA was administered by TDS-IM/EP or by conventional IM injection.
In another study in rabbits, repeated dosing with HIV-MAG with or without GENEVAX® IL-12 given IM/EP as prime followed by two doses of recombinant viral vector boost (Vesicular Stomatitis Virus vaccine [VSV HIV gag]) as boost was also well tolerated.
Preclinical immunogenicity studies in mice and rhesus macaques showed that the HIV-MAG co-administered with IL-12 by IM/EP was able to induce HIV-1 antigen specific cell-mediated immune (CMI) responses as determined by IFN-γ ELISPOT assay. In macaques, each HIV-MAG pDNA construct co-administered with GENEVAX® IL-12 also elicited humoral immune responses measured by ELISA.
Please see the most recent version of the Profectus Biosciences, Inc. (PBS) HIV-MAG -IL12 Investigator's Brochure (Version 2.0, May 2011) for a full description of the preclinical safety profile of this candidate vaccine.

Experience with recombinant human IL-12 pDNA as molecular adjuvant for HIV pDNA vaccines
No clinical studies have yet been conducted testing HIV-MAG vaccine co-administered with GENEVAX® IL-12.
However, other HIV-1 pDNA vaccines expressing antigens similar to HIV-MAG have been tested in Phase 1 clinical studies in combination with GENEVAX® IL-12. The NIH-sponsored clinical trials (HVTN 060, 063, 070) in which different candidate HIV-1 pDNA vaccines coadministered with the GENEVAX® IL-12 given intramuscularly (IM) by needle injection were tested in approximately 250 healthy HIV-uninfected adults. No major safety concerns have been noted in these studies.
In HVTN 080, an HIV-1 pDNA vaccine (PENNVAX™-B gag, pol, env) mixed with GENEVAX® IL-12 was administered IM by in vivo electroporation (EP) using Inovio's Cellectra device. Final safety data are not yet available, but no major safety concerns have been reported. Preliminary

Experience with Ichor TriGrid™Delivery System for in vivo electroporation
To date, the TDS-IM has been utilized as the means of DNA vaccine administration in two completed clinical trials and is currently being evaluated in three ongoing clinical studies. These studies include completed studies of a xenogeneic tyrosinase DNA vaccine candidate in patients with Stage IIB-IV melanoma and a multigenic HIV-1 DNA vaccine candidate (ADVAX) in healthy, HIV uninfected volunteers (IAVI C004). The currently ongoing clinical studies include testing of a multi-epitope malaria DNA vaccine in healthy volunteers, assessment of an epitope based TRP-2 melanoma vaccine in patients with AJCC stage III-IV melanoma, and a multiantigen HIV DNA vaccine administered with or without a DNA based human IL-12 adjuvant in HIV infected individuals. To date, the five trials have enrolled over 90 subjects in electroporation arms of the studies (including subjects receiving either the DNA vaccine candidate or placebo). The device has been used for administration at DNA doses of up to 4.0 mg. Subjects have been administered the vaccine either as a single injection in one muscle site or as two injections in two separate muscle sites. To date, subjects administered the DNA dose as a single injection have received up to five administrations (i.e., five total TDS-IM injections) while subjects administered the DNA dose in two injections have received up to four administrations (i.e., eight total TDS-IM injections).
Adverse events reported in association with use of the device include discomfort/pain during procedure application, minor cutaneous bleeding at the site of injection, and transient injection site soreness of mild to moderate severity, typically resolving within 24-72 hours following administration. Several subjects in the two melanoma studies have reported lightheadedness immediately following procedure application which, in some cases, was accompanied by a decrease in blood pressure. One subject, enrolled in the xenogeneic tyrosinase study, experienced a brief syncopic episode (~30 seconds duration) shortly after procedure application. The subject recovered without incident. At the time of enrollment, the subject indicated a life long history of sinus bradycardia of unknown origin, which was confirmed by electrocardiogram during screening. Multiple electrocardiograms performed after the syncopic episode indicated no changes from pre-procedure baseline. Based on the judgment of the investigator, the subject was withdrawn from the study and the study eligibility criteria modified to exclude subjects with sinus bradycardia. No other serious or unanticipated adverse events attributed to the device or administration procedure have been observed during the five studies.
The results of the completed HIV-1 ADVAX vaccine study in healthy volunteers have been published. 52 Briefly, results from this study indicate that EP based delivery with the TDS-IM device at ADVAX DNA doses ranging from 0.2 -4.0 mg was safe and effective in improving the magnitude, breadth and durability of cellular immune responses to a DNA vaccine candidate. Assessment of the tolerability of the EP procedure by questionnaire after each administration indicates that the procedure is acceptable for healthy, HIV-uninfected volunteers.

Experience with Ad35-GRIN/ENV
Ad35-GRIN/ENV consists of two co-formulated Ad35 vectors: • Ad35-GRIN is a replication-incompetent, recombinant Adenovirus serotype 35 expressing HIV-1 Clade A genes (gag, reverse transcriptase, integrase and nef (GRIN). No related serious adverse event has been reported to date. Reactogenicity events reported seem dose-dependent. Local reactogenicity events are mostly pain and tenderness at the site of injection, mostly mild or moderate in Group A, B, and D volunteers and mild to severe in Group C volunteers. Systemic reactogenicity events reported included chills, fever, malaise, headache, myalgia, and arthralgia. These reactions were predominantly mild or moderate, except in Group C, in which more severe events were observed. All reactogenicity events were transient and resolved spontaneously 53 .
In total, 165 non-serious adverse events have been reported (as of May 5, 2011). The frequency was not dose-related. Two events were assessed as severe (Grade 3): deep vein thrombosis and anxiety disorder; both were considered unrelated to the vaccine. Fifty-eight (58) events were assessed as moderate (Grade 2) in severity: only one was considered possibly related to vaccine (influenza-like illness). One hundred and five (105) events were assessed as mild (Grade 1) in severity. Nine events were considered as possibly related to vaccine (diarrhea, injection site haemorrhage, influenza-like illness, upper respiratory tract injection, 2 nasal congestions, pharyngo-laryngeal pain, pharyngitis, and naso-pharyngitis), one as probably related (injection site anesthesia) and one definitely related (injection site swelling). All other events were considered unrelated or unlikely related to vaccine.
Moderate or greater abnormal clinical laboratory values were observed in five volunteers: elevated AST in two subjects, low haemoglobin in two women (all moderate and not considered as related to vaccination), and elevated ALT in one subject (severe and not considered as related to vaccination).

Safety and tolerability:
To assess safety and tolerability of the different prime-boost regimens: 1. Proportion of volunteers with moderate or greater reactogenicity (i.e., solicited adverse events) during a 7 day follow-up period after each vaccination 2. Proportion of volunteers with moderate or greater and/or vaccine-related unsolicited adverse events (AEs) including safety laboratory (biochemical, haematological) parameters, from the day of each vaccination up to 28 days post each vaccination.
3. Proportion of volunteers with vaccine related serious adverse events (SAEs) collected throughout the study period IAVI Protocol B004, Version 1.0 Page 21 of 79 30Jun11

Secondary Endpoints
Immunogenicity: To assess (qualitative and quantitative) immune responses elicited by the different prime-boost regimens: 1

Exploratory Endpoints
Immunogenicity: Additional immunogenicity assessments may include: 1. Binding antibodies to HIV antigens (frequency and magnitude) 2. Neutralizing antibodies to HIV antigens (seropositivity rates and magnitude of antibody titers)

Neutralizing antibodies to the Ad35 vector (frequency and magnitude)
Responses may be further characterized to include: 1. Ability of CD8 cells to inhibit HIV replication in a Viral Inhibition Assay (VIA)

Capacity of antigen specific T cells to proliferate
3. Flow cytometry to assess markers for memory, exhaustion and activation, and secretion of cytokines other than IFN-γ, IL-2 and TNF-α

Repertoire analysis to assess B cell response
Serum antibodies against human IL-12:

Proportion of volunteers/vaccine recipients who develop antibodies against human IL-12
EP Tolerability: 1. Proportion of volunteers judging the procedure as acceptable

Study Design
The study is a randomized, double-blind placebo-controlled trial.

Duration of the Study
Volunteers will be screened up to 42 days before vaccination and will be followed for 12 months after the initial vaccination. It will take approximately 3 months to enrol 75 volunteers. The anticipated study duration for each volunteer is approximately 13 months from screening through last study visit.

Study Population
The study population consists of healthy male or female adults aged 18-50 years at low risk for HIV infection, who are willing to undergo HIV testing, use an effective method of contraception, and who in the opinion of the investigator or designee, understand the study and provide written informed consent.
Approximately 75 volunteers (60 vaccine recipients, 15 placebo recipients) who meet all eligibility criteria will be included in the study. An over-enrolment of up to 10% (up to 83 volunteers total) will be permitted in the study to facilitate rapid enrolment.

Inclusion Criteria
1. Healthy male or female, as assessed by a medical history, physical exam, and laboratory tests; 2. At least 18 years of age on the day of screening and has not reached his/her 51 st birthday on the day of first vaccination; 3. Willing to comply with the requirements of the protocol and available for follow-up for the planned duration of the study; 4. In the opinion of the Principal Investigator or designee, and based on Assessment of Informed Consent Understanding (AOU) results, has understood the information provided and potential risks linked to vaccination and participation in the trial; written informed consent will be provided by the volunteer before any study-related procedures are performed; 5. Willing to undergo HIV testing, risk reduction counselling, receive HIV test results and committed to maintaining low risk behaviour for the trial duration; 6. If a female of childbearing potential, willing to use an effective non-barrier method of contraception (hormonal contraceptive or intrauterine device [IUD]) from screening until at least 4 months after the last study vaccination; 7. Assessed by the clinic staff as being at "low risk" for HIV infection on the basis of self-reported sexual behaviour within the 12 months prior to enrolment defined as follows: a. Sexually abstinent, or b. Had two or fewer mutually monogamous relationships with partners who did not use illicit drugs, or c. Had two or fewer partners believed to be HIV-uninfected and who did not use illicit drugs (illicit drug use or abuse that includes any injection drugs, methamphetamines [crystal meth], heroin, cocaine, including crack cocaine or chronic marijuana abuse) and with whom he/she regularly used condoms for vaginal and anal intercourse; 8. All female volunteers must be willing to undergo urine pregnancy tests at time points indicated in the Schedule of Procedures (Appendices A, B) and must test negative prior to each study vaccination; 9. All sexually active males (unless anatomically sterile or in a monogamous relationship with a female partner who uses a documented non-barrier method of birth control) must be willing to use an effective method of contraception (such as consistent condom use) from the day of first vaccination until at least 4 months after the last vaccination; 10. Willing to forgo donations of blood or any other tissues during the study and, for those who test HIV-positive due to trial vaccination (vaccine-induced HIV seropositivity), until the anti-HIV antibody titres become undetectable.

Exclusion Criteria
1. Confirmed HIV-1 or HIV-2 infection; 2. Any clinically relevant abnormality on history or examination including history of immunodeficiency or autoimmune disease; use of systemic corticosteroids (the use of topical or inhaled steroids is permitted); immunosuppressive, anti-cancer, anti-tuberculosis or other medications considered significant by the investigator within the previous 6 months; 3. Any clinically significant acute or chronic medical condition that is considered progressive, or in the opinion of the investigator, makes the volunteer unsuitable IAVI Protocol B004, Version 1.0 Page 24 of 79 30Jun11 for participation in the study; 4. Reported risky behaviour for HIV infection within 12 months prior to vaccination, as defined by: • Unprotected sexual intercourse with a known HIV-infected person, a partner known to be at high risk of HIV infection or a casual partner (i.e., no continuing established relationship) • Engaged in sex work • Frequent excessive daily alcohol use or frequent binge drinking or chronic marijuana or any other use of use of illicit drugs • History of newly-acquired syphilis, gonorrhoea, non-gonococcal urethritis, HSV-2, chlamydia, pelvic inflammatory disease (PID), trichomonas, mucopurulent cervicitis, epididymitis, proctitis, lymphogranuloma venereum, chancroid, or hepatitis B; • Three or more sexual partners 5. If female, pregnant or planning a pregnancy within 4 months after last study vaccination; or lactating; 6. Asthma requiring high-dose oral or inhaled corticosteroids; 7. Bleeding disorder that was diagnosed by a physician (e.g., factor deficiency, coagulopathy or platelet disorder that requires special precautions) (Note: A volunteer who states that he or she has easy bruising or bleeding, but does not have a formal diagnosis and has IM injections and blood draws without any adverse experience, is eligible); 8. History of splenectomy; 9. Any of the following abnormal laboratory parameters listed below: Haematology

Recruitment of Volunteers
Healthy adult male and female volunteers may be recruited through information presented in community organizations, hospitals, colleges, other institutions and/or advertisements to the general public. This information will contain contact details.

Screening Period
During Screening, study staff will perform the following procedures: Screening laboratory test(s) may be repeated once at the discretion of the Principal Investigator or designee to investigate any isolated abnormalities.
If the screening visit occurs more than 42 days prior to the date of 1 st vaccination, all screening procedures must be repeated. The complete medical history may be replaced by an interim medical history and the Informed Consent form/ Volunteer Information Sheet should be reviewed.
If a volunteer has signed the consent form, but does not meet the eligibility criteria, the records must be kept at the site.

Vaccination Visits
Prior to the first vaccination, study staff will: • Answer any questions about the study Study staff will observe volunteers closely for at least 30 minutes after each vaccination for any acute reactogenicity. At the end of the observation period study staff will: • Record vital signs (pulse, respiratory rate, blood pressure and temperature) • Assess any local and systemic reactogenicity • Assess any other adverse events • Ask volunteer to complete EP tolerability assessment (unless volunteer is in Group 5, in which case they will complete the EP tolerability assessment following their Month 4 vaccination only)

Subsequent Vaccination Visits:
Study staff will perform the same procedures as above with the following exceptions: • Review the routine safety laboratory parameters (Section 9.1.6), as appropriate, from the previous visit prior to each vaccination. If a volunteer has an abnormal laboratory value that is known at the time of vaccination, follow the specified guidelines (Section 12.0) • Conduct pre HIV-test counselling if an HIV test is scheduled (see Appendices A-B) or clinically indicated • Provide post-test counselling if the results of a prior HIV test are being communicated to the volunteer • Do not conduct EP tolerability assessment following vaccinations that do not use EP

Post-Vaccination Visits
The volunteer will be asked to maintain a Memory Aid from the day of each vaccination and for the next 7 days. Study staff will review the Memory Aid with the volunteer, determine the severity of the reactions through volunteer discussion and record the information on applicable source documentation. There will be scheduled clinic visits 3, 7 and 14 days after each vaccination for an assessment by clinic staff.
The following procedures will be conducted at these visits: • Collect concomitant medication information • If any signs or symptoms are present, perform a symptom-directed physical examination • Assess any adverse events, including local and systemic reactogenicity • Collect specimens for all tests as indicated in the Schedule of Procedures (Appendices A-B)

Additional Follow-up Visits
Assessments and procedures will be performed according to the Schedule of Procedures (Appendices A-B).

Unscheduled Visits
Unscheduled Visits/Contacts are visits/contacts that are not described in the Schedule of Procedures (Appendices A-B). Unscheduled visits may occur any time during the study: • For administrative reasons, e.g., the volunteer may have questions for study staff or may need to re-schedule a follow-up visit • To obtain laboratory test results from a previous visit • For other reasons as requested by the volunteer or site investigator IAVI Protocol B004, Version 1.0 Page 28 of 79 30Jun11 All unscheduled visits will be documented in the volunteer's study records and on applicable source documents and entered into the study database.

Final Study Visit or Early Termination Visit
Assessments and procedures will be performed according to the Schedule of Procedures (Appendices A-B).

Informed Consent Process
A sample Informed Consent Document consisting of a Volunteer Information Sheet and a Consent Form is provided by the Sponsor to the CRC. This document is made sitespecific and translated (if necessary), submitted and approved by the Independent Ethics Committee (IEC)/ Ethics Review Board (ERB).

Volunteer Information Sheet/ Informed Consent Document
A qualified member of the study staff will conduct the informed consent process by reviewing the Volunteer Information Sheet.
The following study-specific elements are included: 1. The vaccines tested in this study cannot cause HIV or AIDS.
2. If a vaccine recipient is exposed to HIV through exposure in the community and acquires HIV, it is not known whether the study vaccine(s) could increase, decrease or have no effect on: a) the risk of becoming infected with HIV; b) if infected, the course of HIV infection and; c) if infected, the time it takes to develop AIDS after being infected 3. The vaccine recipient may develop antibodies against HIV following vaccination, which is desirable but may produce a positive result in a routine HIV antibody test, and that provisions have been made to distinguish between response to vaccine and natural HIV infection during and after the study. In case the volunteer has a positive result due to vaccine-induced antibodies in a routine HIV antibody test, he/she will be followed until the result is no longer positive. 4. Women of childbearing potential should use a reliable non-barrier form of contraception from screening, during the vaccination period and until 4 months after the last vaccination 5. Placebo will be administered in this study and volunteer may receive placebo throughout the study

Consent Form
All volunteers will give their written informed consent to participate in the study on the basis of appropriate information and with adequate time to consider this information and IAVI Protocol B004, Version 1.0 Page 29 of 79 30Jun11 ask questions. To confirm that the volunteer has understood the information contained in the Volunteer Information Sheet, an Assessment of Informed Consent Understanding (AOU) will be administered, as per site-specific procedures (see SOM).
The volunteer's consent to participate must be obtained by him/her signing (or if illiterate, marking) and dating the Informed Consent Form. The person obtaining consent will also sign. If the volunteer is functionally illiterate, the complete Informed Consent Document (which includes the Volunteer Information Sheet) must be read to him/her in the language that he/she best understands in the presence of an independent literate observer not affiliated with the study, who will sign and date the consent form as an impartial witness.
The signed/marked and dated Informed Consent Document must remain at the study site. A copy of the signed/marked and dated Informed Consent Document will be offered to the volunteer to take home. Those volunteers who do not wish to take a copy will be required to document that they declined to do so.
Family members, sexual partner(s) or spouse(s) will be offered education and counselling regarding a volunteer's participation in the study ONLY if the participating volunteer has specifically given written consent for such education and counselling.

Medical History and Physical Examination
At screening, a comprehensive medical history will be collected, including details of any previous vaccinations and reaction to vaccinations, history of STIs and contraceptive practices. At subsequent visits, an interim medical history will be performed.
A general physical examination includes height, weight, examination of skin, respiratory, cardiovascular and abdominal systems, an assessment of cervical and axillary lymph nodes and recording of vital signs (pulse, respiratory rate, blood pressure and temperature). Skin fold thickness (Skin Pinch Test) will be measured at the first physical exam.
A symptom-directed physical examination includes assessment of cervical and axillary lymph nodes, recording of vital signs (pulse, respiratory rate, blood pressure and temperature) and any further examination indicated by history or observation.

HIV Testing and HIV-test Counselling
Additionally, study staff will perform pre-HIV test counselling (prior to collecting blood for an HIV test) and post-HIV test counselling (when HIV test results are available) according to the Schedule of Procedures (Appendices A-B). For more information on HIV testing and HIV-test counselling, see Section 11.0.

HIV Risk Reduction Counselling
Study staff will provide HIV risk reduction counselling based on reported individual risk and provide free condoms, as appropriate, at every visit. The procedures for risk reduction counselling will be detailed in site-specific SOPs.

Family Planning Counselling
Study staff will counsel volunteers about the importance of preventing pregnancies and use of condoms, as well as other effective family planning methods, as appropriate. Free condoms are provided and volunteers may be referred for family planning services either on-site or to a family planning clinic, as necessary and according to site-specific SOPs. Contraceptive methods chosen and compliance will be documented.

Specimens
Up to approximately 103 mL of blood will be collected at visits, usually from the antecubital fossa, according to the Schedule of Procedures (Appendices A-B).
All specimens will be handled according to the procedures specified in the Study Operations Manual (SOM) and Laboratory Analytical Plan (AP).
In the event of an abnormal laboratory value, volunteers may be asked to have an additional sample collected at the discretion of the Principal Investigator or designee.

Reimbursement
Volunteers will be reimbursed for their time, effort and for costs to cover their travel expenses to the study site and any inconvenience caused due to study participation. Reimbursement will be made after the completion of each study visit. Site-specific reimbursement amounts will be documented in the site-specific Volunteer Information Sheet and approved by the Ethics Committee.

Randomization and Blinding
Volunteers will be identified by a unique volunteer identification number.
Volunteers will be randomized across all groups. The randomization schedule will be prepared by the statisticians at the Data Coordinating Centre (DCC) prior to the start of the study. Volunteers will be automatically assigned a specific allocation number as they are enrolled into the data entry system. An unblinding list will be provided to the unblinded site pharmacist by the DCC.
Study staff and volunteers will be blinded to vaccine versus placebo and dosage levels of GENEVAX® IL-12, but not to schedule, number of vaccinations administered and delivery method.
A volunteer will be considered enrolled once she/he has been randomly allocated to a specific vaccination regimen.
Enrolment will be staggered with 1 volunteer across all CRCs enrolled per day for the first 4 volunteers.
Volunteers will be informed about their assignment (vaccine/placebo) at study completion, once the database is locked. Should a study volunteer be unblinded during the study, further administration of the Investigational Product (vaccine or placebo) will be discontinued. The study volunteer will be followed up until the end of the study according to Schedule of Procedures (Appendices A-B).

Unblinding Procedure for Individual Volunteers
Unblinding of an individual volunteer may be indicated in the event of a medical emergency if the clinical management of the volunteer would be altered by knowledge of the treatment assignment.
The unblinding information should be restricted to a small group of individuals involved in clinical management of the volunteer (e.g., treating physician) and maintain the blind for those responsible for the study assessments.
The reasons for unblinding should be documented and the IAVI Chief Medical Officer, the Medical Monitor and the DCC should be notified as soon as possible. The procedures and contact numbers for unblinding are outlined in the Study Operations Manual (SOM).

Referral to Long Term Follow-Up Study
To assess the long-term safety of the Investigational Products, study volunteers will, after completion of all visits in this study, be offered participation in a long-term follow-up study for approximately 5 years following the last study injection. This study includes a health assessment questionnaire and HIV testing. Additional blood samples may be collected to assess the persistence of the immune responses in vaccine recipients. A separate informed consent will be administered for this long-term follow-up study. o The Ad35-GRIN/ENV vaccine is manufactured by Transgene (France) and supplied by IAVI.
o Sodium Chloride Injection, USP 0.9% will be used as placebo for the HIV-MAG and GENEVAX® IL-12 and Ad35 vaccines.
o The TDS-IM electroporation device is manufactured by California MedTech (US) and Life Science Outsourcing (US) and supplied by Ichor Medical Systems, Inc.

HIV-MAG
The The individual vaccine plasmids are supplied at 3.0 mg/mL in 30 mM citrate buffer pH 6.5 containing 0.15 M NaCl, 0.01% EDTA, and 0.25% bupivacaine-HCl in a 2 mL container with a Daikyo/West Plug stopper and aluminium flip seal. The filling volume per container is 0.8 mL±0.04 mL.

Ad35-GRIN/ENV
Ad35-GRIN/ENV consists of two vectors Ad35-GRIN and Ad35-ENV formulated in a 1:1 ratio and filled into single use vials for intramuscular injection.
• Ad35-GRIN is a recombinant replication-incompetent Adenovirus serotype 35 expressing HIV-1 subtype A gag, reverse transcriptase, integrase, and nef genes. Ad35-GRIN/ENV is supplied as a frozen sterile formulation in a 4-mL vial with a butyl stopper and aluminum seal. Each vial contains 0.725 mL of vaccine. The volume of administration is 0.5 mL, which will deliver a final dosage of 2x10 10 vp per dose. The dose of the vaccine is provided as a total virus particle count measured by HPLC and expressed as viral particle (vp). The vaccine is formulated in buffer composed of Tris 10 mM pH 8.5, Sucrose 342,3 g/L, 1mM MgCl 2 , Tween80 54 mg/L and 150 mM NaCl in water for injection (used for diluting the purified bulk). Vaccine is a whitish liquid and limpid or slightly turbid liquid, depending on the virus concentration.

GENEVAX® IL-12
GENEVAX® IL-12 will be provided at 2.0 mg/mL and is formulated in 30 mM citrate buffer (pH 6.5) containing 150 mM NaCl, 0.01% EDTA, and 0.25% bupivacaine-HCl in a 2mL container with Daikyo/West Plug Stopper and aluminium flip seal. The volume per container is 0.9 mL ± 0.04 mL.

Placebo
Commercially available Sodium Chloride Injection, USP 0.9% will be used as placebo.
The summary of the Investigational Products is shown in

. Ichor Medical Systems TriGrid™ Delivery System (TDS-IM)
The TDS-IM is an electroporation based delivery device designed for IM administration of DNA. Specifically, the device is designed to propagate EP-inducing electrical fields at the site of administration in the presence of the DNA to be delivered. It consists of three components which include a single use Application Cartridge, an Integrated Applicator, and a Pulse Stimulator. The device is a designated Class II medical device for investigational use.

Application Cartridge
The Application Cartridge is used to house the agent to be delivered (in a standard syringe) and the electrodes used for EP application. Each Application Cartridge is packaged sterile for single use and is the only subject contact component of the system. It is composed of a plastic, injection molded body that encloses four electrodes arranged to form two equilateral triangles with an adjoining base.
Prior to the administration procedure, the Application Cartridge is removed from the sterile pouch. The agent to be administered is loaded into a standard Becton To accommodate differences in skin thickness, a slidable depth control gauge allows adjustment of injection depth to one of three settings: 12, 17, or 22 millimeters from the skin surface. Prior to device activation, the electrodes and injection needle remain recessed within the sterile cartridge body. A plastic safety cap located on the tip of the cartridge protects the operator from accidental stick injury and ensures that the electrodes remain sterile prior to administration.

Integrated Applicator
The Integrated Applicator is a reusable, hand-held electromechanical device that contains mechanisms to deploy the electrodes and injection needle into the target muscle tissue and administer the agent of interest. The device is configured so that activation of the device allows the entire procedure to be applied in an automated fashion. As a result, the administration parameters (including the site of agent injection relative to EP application, rate of agent injection, and time interval between agent injection and EP application) will be implemented in a uniform fashion for each subject.

Pulse Stimulator
The Integrated Applicator is connected to the Pulse Stimulator through an incorporated cable. The Pulse Stimulator controls the administration sequence, generates the electrical signals necessary to enhance the intracellular delivery of the agent, and monitors the administration sequence for safety hazards. It is an electronic device that is compatible with 120 V/60Hz or 240 V/50Hz supply. The EP conditions used in this study will be applied at an amplitude of 200 V and a total duration of 40 mS, with a 10% duty cycle (e.g., 40 mS of active voltage application within a 400 mS period). The Pulse Stimulator performs a comprehensive self diagnostic upon start-up to ensure that the device and all internal safety systems are functioning properly before an administration procedure can be initiated. In the event of a problem, the user will be notified by the display of an error code on the digital display located on the front of the unit.

Shipment and Storage
Authorization to ship the Investigational Products to the CRC will be provided in writing by the Sponsor, upon confirmation that all required critical documents for shipment authorization are completed. The Investigational Products will be shipped maintaining the required storage conditions and stored in a secure location in the clinical site's pharmacy.
HIV-MAG will be stored at 2-8 o C GENEVAX® IL-12 will be stored 2-8 o C Ad35-GRIN/ENV will be stored at -70°C or below Sodium Chloride Injection, USP 0.9% (placebo) will be stored at 20-25 o C TDS-IM device components will be stored at room temperature Each of the investigational products will have compliant primary labels affixed onto the vials that will contain unique lot numbers, storage temperature and a US cautionary statement.

Preparation of Investigational Product (IP)
Detailed instruction will be provided to the pharmacist for preparing each of the investigational products. The pharmacist will not be blinded, but the study physician administering the vaccine will be blinded. Injections should be given within 4 hours of preparation. Final volume for administration is in Table 8.1.4.-1. Instructions for storing used vials until the end of the study and subsequent disposal will be provided. Syringes or other components in direct contact with investigational products will be disposed of in a biohazard container and incinerated or autoclaved.
For details of preparation of vaccines, please see Pharmacy Instructions in the SOM.

Administration of Investigational Product
Investigational Product will be administered according to the Schedule of Procedures (Appendices A-B).
All administrations of HIV-MAG with or without GENEVAX® IL-12 or the respective placebo consist of 2 IM injections, one into each medial deltoid, by in vivo electroporation (IM/EP) using the Ichor Medical Systems TriGrid™ Delivery System (TDS-IM). Prior to injection, a skin pinch test will be performed to set the dept adjuster. Details are provided in the SOM.
The preferred site of administration of Ad35-GRIN/ENV is the deltoid muscle of the nondominant upper arm (for example, injection in the left arm if the volunteer uses mainly the right arm), unless contraindicated for another reason.
Further information on the administration of the Investigational Products is supplied in the SOM.

Accountability and Disposal of Investigational Product
The unblinded study pharmacist at each site will be responsible for vaccine accountability and preparation. A witness will be required to confirm accuracy of IP preparation. Verification and sign-off sheets will need to be completed and signed-off for each IP preparation by the pharmacist and counter-signed by the witness.
All used vials will be returned to the pharmacy at the end of each vaccination visit. The date, vial allocation number and location of storage of the returned vials will be recorded. The TDS-IM cartridges with the injection needles affixed inside will be placed in a sharps container following delivery.
During the study, the Investigational Product accountability form, the dispensing log and the log of returned vials will be kept and monitored.
At the end of the study, the used and unused vials will be destroyed; destruction will be witnessed, according to IAVI and site-specific SOPs.

Safety Assessments
Data on local and systemic reactogenicity (i.e., solicited AEs) will be collected by structured interview and medical examination. Volunteers will be given a Memory Aid, IAVI Protocol B004, Version 1.0 Page 36 of 79 30Jun11 which is a tool to assist with collecting reactogenicity data, but will not be saved as a source document. Data on other adverse events will be collected with open-ended questions. All data will be recorded on the appropriate source documents and entered into the study database.
Local and systemic reactogenicity events will be solicited by study staff prior to vaccination and at least 30 minutes post-vaccination, as specified in the Schedule of Procedures (Appendices A-B).

Local reactogenicity
The presence of local reactogenicity will be assessed at the time points specified in the Schedule of Procedures (Appendices A-B).
Pain, tenderness, erythema/skin discoloration, swelling/hardening or thickening will be assessed and graded using Appendix C, Adverse Event Severity Assessment Table, as a guideline.

Systemic reactogenicity
The presence of systemic reactogenicity will be assessed at the time points specified in the Schedule of Procedures (Appendices A-B).
Fever, chills, headache, nausea, vomiting, malaise, arthralgia, myalgia and fatigue will be assessed and graded using Appendix C, Adverse Event Severity Assessment Table, as a guideline.

Vital signs
At the vaccination visits, vital signs (pulse, respiratory rate, blood pressure and temperature) will be measured by study staff prior to vaccination and at least 30 minutes post-vaccination. At the other study visits, vital signs will be assessed at the time points specified in the Schedule of Procedures (Appendices A-B).

Other adverse events
Other adverse events (AEs) will be collected through one month after the last vaccination in all groups. Serious Adverse Events (SAEs) will be collected throughout the entire study period. Open ended questions will be asked at time points according to the Schedule of Procedures (Appendices A-B). All adverse events will be graded using Appendix C, Adverse Event Severity Assessment Table, as a guideline and will be assessed for causality to the IP.
For more information regarding adverse events refer to Section 10.0, Adverse Events.

Concomitant Medications
During the study, information regarding concomitant medications and reasons for their use will be solicited from the study volunteers at each visit and recorded. If clinically indicated, inactivated/killed/subunit vaccines (non-HIV) and immunoglobulin may be given up to 14 days before study vaccination(s) or 14 days after the most recent study vaccination (following the post-vaccination blood draw). Live-attenuated vaccines may be given 60 days before study vaccination(s) or 60 days after the most recent post-vaccination blood draw. However, the study vaccination(s) should not be given if there are any continuing symptoms from recently administered non-study vaccines. In this situation, the site investigator should consult with the Medical Monitor before administering the next study vaccination.
If the use of a short tapering course (<2 weeks) of oral corticosteroids is required, the study vaccinations may be continued after a 4-week washout period provided that the medical condition requiring this therapy has completely resolved and in the opinion of both, the site investigator and Medical Monitor, the continuation of the study vaccinations will not jeopardize the safety of the volunteer. Volunteers requiring chronic (> 2 weeks) or long term therapy will not receive any further vaccinations but will continue with follow-up visits until the end of the study. Table 9.1.6-1 shows the laboratory parameters that will be measured routinely. These parameters will include haematology, clinical chemistry, immunological assays and urinalysis. The samples for these tests will be collected at the time points indicated in the Schedule of Procedures (Appendices A-B).

Antibody Responses
• Antibodies against HIV proteins will be measured according to time points as indicated on the Schedule of Procedures (Appendices A-B).
Binding antibodies to HIV antigens (frequency and magnitude) Neutralizing antibodies to HIV antigens (seropositivity rates and magnitude of antibody titers) Neutralizing antibodies to the Ad35 vector (frequency and magnitude) • Other functional antibody assays (e.g., ADCC, ADCVI) may be performed at any of the pre-determined time points

Cellular Responses
Immunogenicity assays, including ELISPOT and intracellular cytokine staining (ICS) for monitoring the number of circulating T cells that can be stimulated to produce cytokines and other effector molecules, will be performed at time points indicated in the Schedule of Procedures (Appendices A-B), using peptide pools representing all or a portion of the encoded HIV antigens.
Further exploratory studies will be carried out to look at the breadth of the response via epitope mapping. Vaccine-matched or other HIV peptide sets such as PTE peptides will be used as the first screen. For examination of crossreactive T cell responses peptides from HIV-subtypes C and D may be used. Vaccine-induced T cell responses may be further characterized for HLA restriction and additional markers on the responding cells, such as markers for memory, activation, function or markers for homing to mucosal tissues. The ability of PBMC to restrict the growth of HIV in vitro may be examined using a Virus Inhibition Assay (VIA). The analytical plan for the immunology studies will be developed and an algorithm will be applied to determine which samples and time points are prioritized for exploratory assays.
Additional cellular, humoral and other immunologic assays may be conducted to assess HIV and vector-specific responses.

PBMC, Serum and Plasma Storage
Samples of cryopreserved PBMC, plasma and serum will be stored as indicated in the Schedule of Procedures (Appendices A-B) and may be used for the purposes of standardisation, quality control and for future assays related to HIV vaccine research and development. These samples will be archived and the testing laboratories will be blinded to the volunteer's identity.

9.3
Other Assessments

HLA Typing
Samples for HLA typing will be collected as specified in the Schedule of Procedures (Appendices A-B) and may be analyzed as warranted.

Serum Antibodies against Human IL-12
Samples for anti-IL12 antibodies will be collected as specified in the Schedule of Procedures (Appendices A-B) and may be analyzed as warranted.

HIV test
Samples will be tested at the time points indicated in the Schedule of Procedures (Appendices A-B). Further information is specified in Section 11.1 HIV Testing.

Pregnancy Test
A urine pregnancy test for all female volunteers will be performed by measurement of Human Chorionic Gonadotrophin (βHCG) at time points indicated in the Schedule of Procedures (Appendices A-B). The results of the pregnancy test must be available and negative prior to each vaccination.

Screening Assessment
A Screening Assessment will be administered at the Screening Visit.

HIV Risk Assessment
Study staff will assess volunteers for their past and current risk of acquiring HIV at time points indicated in Schedule of Procedures (Appendices A and B)

Electroporation Tolerability Assessment
An Electroporation Tolerability Assessment Tool will be provided to the volunteers at each EP administration time point to complete immediately after the procedure, as indicated in Schedule of Procedures (Appendices A and B)

Social Impact Assessment
Each volunteer will have a social impact assessment administered at the time points specified in the Schedule of Procedures (Appendices A and B). This assessement is intended to assess the impact of trial participation on the volunteer's daily life, if any.

Definition
An adverse event (AE) is any untoward medical occurrence in a volunteer administered Investigational Product and which does not necessarily have a causal relationship with the Investigational Product. An AE can therefore be any unfavourable or unintended sign (including an abnormal laboratory finding), symptom, or disease, temporally associated with the use of the Investigational Product whether or not related to the Investigational Product.

Assessment of Severity of Adverse Events
Assessment of severity of all AEs, including SAEs, is ultimately the responsibility of the Principal Investigator of each site.
The following general criteria should be used in assessing adverse events as mild, moderate, severe or very severe at the time of evaluation: Guidelines for assessing the severity of specific adverse events and laboratory abnormalities are listed in Appendix C, Adverse Event Severity Assessment Table.

Relationship to Investigational Product
The relationship of an (S)AE to Investigational Product (IP) is assessed and determined by the Principal Investigator or designee. All medically indicated and available diagnostic methods (e.g., laboratory, blood smear, culture, X-ray, etc…) should be used to assess the nature and cause of the AE/SAE. Best clinical and scientific judgment should be used to assess relationship of AE/SAEs to the IP and/or other cause.
The following should be considered: • Presence/absence of a clear temporal (time) sequence between administration of the Investigational Product and the onset of AE/SAE • Presence/absence of another cause that could more likely explain the AE/SAE (concurrent disease, concomitant medication, environmental or toxic factors, etc.) • Whether or not the AE/SAE follows a known response pattern associated with the Investigational Product The relationship should be reported as one of the following: Possibly: equally likely explained by another cause but the possibility of the Investigational Product relationship cannot be ruled out (e.g., reasonably well temporally related and/or follows a known Investigational Product response pattern but equally well explained by another cause).
Probably: more likely explained by the Investigational Product (e.g., reasonably well temporally related and/or follows a known Investigational Product response pattern and less likely explained by another cause).
Definitely: clearly related and most likely explained by the Investigational Product.
For the purpose of expedited safety reporting, all possibly, probably or definitely related SAEs are considered Investigational Product-related SAEs.

An adverse event is reported as a "Serious Adverse Event" if it meets any the following criteria (as per International Conference on Harmonisation [ICH] Good Clinical Practice [GCP] Guidelines):
• Results in death • Is life threatening • Results in persistent or significant disability/incapacity • Requires in-patient hospitalization or prolongs existing hospitalization • Is a congenital anomaly/birth defect or spontaneous abortion • Any other important medical condition that requires medical or surgical intervention to prevent permanent impairment of a body function or structure Serious Adverse Events (SAEs) should be reported to IAVI within 24 hours of the site becoming aware of the event. All SAEs should be reported using the designated SAE Report Form and sent to the Sponsor as described in the SOM.
To discuss Investigational Product-related SAEs or any urgent medical questions related to the SAE, the site investigator should contact one of the IAVI medical monitors directly (see the Contact List).
The IAVI SAE Report Form should be completed with all the available information at the time of reporting. The minimum data required in reporting an SAE are the volunteer identification number, date of birth, gender, event description (in as much detail as is known at the time), onset date of event (if available), reason event is classified as serious, reporting source (name of Principal Investigator or designee) and relationship to the Investigational Product as assessed by the investigator.
The Principal Investigator or designee is required to write a detailed written report with follow up until resolution or until it is judged by the Principal Investigator or designee to have stabilized. The Principal Investigator or designee must notify the local IRB/IEC of all SAEs as appropriate.
In case of Investigational Product-related SAEs, the Sponsor will notify responsible regulatory authorities, Safety Review Board (SRB), and other study sites where the same Investigational Product is being tested.
More details on SAE definitions and reporting requirements are provided in the SOM.

Clinical Management of Adverse Events
Adverse events (AEs) will be managed by the clinical study team who will assess and treat the volunteer as appropriate, including referral. If any treatment/medical care is required as a result of harm caused by the Investigational Product or study procedures, this will be provided free of charge.
If a volunteer has an AE and/or abnormal laboratory value that is known at the time of study vaccination, the specifications of Section 12.0 will be followed.
Volunteers will be followed until the AE resolves or stabilizes or up to the end of the study, whichever comes last. If at the end of the study, an AE (including clinically significant laboratory abnormality) that is considered possibly, probably or definitely related to the Investigational Product is unresolved, follow-up will continue until resolution if possible and the volunteer will be referred.

Pregnancy
Although not considered an AE, if a female volunteer becomes pregnant during the study, it is the responsibility of the Principal Investigator or designee to report the pregnancy promptly to IAVI using the designated forms. Vaccinations will be discontinued and the volunteer followed for safety until the end of pregnancy or study completion, whichever occurs last.
Complications of pregnancy that meet criteria for serious specified in Section 10.4 of this Protocol (e.g., eclampsia, spontaneous abortion, etc.) should be reported as SAEs.
If a female volunteer becomes pregnant during the study, then, if possible, approximately 2-4 weeks after delivery, the baby will be examined by a physician to assess its health status and the results will be reported to IAVI.

Intercurrent HIV Infection
HIV infection cannot be caused by the Investigational Products. If a volunteer acquires HIV through exposure in the community, study vaccinations must be discontinued, and the volunteer should have an Early Termination (ET) visit and offered referral to Protocol H, as described in Section 11.3.2.
Intercurrent HIV infection in study volunteers, although not considered an SAE,must be reported promptly to IAVI using the designated forms. However, medical conditions associated with the HIV infection that meet criteria for serious specified in the Section 10.4 of this Protocol (e.g., sepsis, PCP pneumonia, etc.) should be reported as SAEs using the SAE Report Form.

Serious Event Prior to Investigational Product Administration
If a serious event occurs in the period between the volunteer signing the Informed Consent Form and receiving the first study vaccination, the event will be reported using the SAE form and following the same procedures for SAE reporting, as indicated in Section 10.4. The timing of the event will be indicated by using the relevant checkbox on the SAE form.

HIV Testing
All Volunteers will be tested for HIV antibodies as indicated in the Schedule of Procedures (Appendices A-B) or as needed, if medical or social circumstances arise. All volunteers will receive HIV risk reduction counselling and pre-HIV-test and post-HIV-test counselling, as specified in Section 11.3.2 Counselling.
A site-specific, predetermined HIV testing algorithm will be followed. In case the routine HIV antibody test is positive, this algorithm will differentiate between an immune response to the vaccine(s) and acquisition of HIV through exposure in the community.
Volunteers who have (a) positive HIV-antibody test(s) as a result of vaccine-induced HIV antibodies will have their test results reported as "Not infected with HIV-1 or HIV-2" (to prevent unblinding of volunteer and staff). A vaccine recipient who still tests HIV positive at the end of the study will be informed of his/her positive test result and offered continuing follow-up until the test becomes negative. If a volunteer is found to be HIV-infected, a newly drawn blood specimen will be collected for confirmation.
Should a volunteer require an HIV test outside the study for personal reasons, it is recommended that the volunteer contact the study staff first. The HIV test can be done at the study site and then processed at an independent laboratory as above. Written evidence of HIV status (HIV-infected or HIV-uninfected) will be provided upon request.

Social Discrimination as a Result of an Antibody Response to Vaccine
In order to minimize the possibility of social discrimination in volunteers (if any) who develop vaccine-induced HIV antibodies and test positive on a diagnostic HIV antibody test, appropriate diagnostic HIV testing and certification will be provided both during and after the study as needed.

HIV Infection
Volunteers who are found to be HIV infected at screening (prevalent HIV-infection) and volunteers who acquire HIV infection during the study (intercurrent HIV-infection) will be provided the following:

Counselling
The volunteer will be counselled by the study counsellors. The counselling process will assist the volunteer with the following issues:

Referral for Support, Care and Treatment
Volunteers will be referred to a patient support centre or institution of his/her choice for a full discussion of the clinical aspects of HIV infection. Referral will be made to a designated physician or centre for discussion of options of treatment of HIV-infection.
For those individuals who become HIV infected after enrolment in the study (i.e., from first vaccination through final study visit), antiretroviral therapy will be provided when clinically indicated according to accepted treatment guidelines. According to IAVI's Treatment and Care Guidelines, antiretroviral therapy will be provided at no charge for up to 5 years after treatment is initiated, if it is not available through another program.
Volunteers with confirmed HIV infection should have an early termination visit and be asked for their consent to participate in Protocol H. Ideally, the Early Termination (ET) visit and Protocol H study entry visit should occur on the same day.
In the unlikely event that HIV infection is confirmed during a reactogenicity period, contact the medical monitor to determine the course of action on a case by case basis.
HIV-infected pregnant women will be referred for prenatal care and to a program for the Prevention of Mother to Child Transmission (PMTCT) as per site-specific procedures. The pregnant volunteer will be followed according to timeline as specified in Section 10.6.

Discontinuation of Vaccinations
Any discontinuation or planned discontinuation from further vaccinations will be discussed with the Sponsor. Volunteers will be discontinued from further vaccination for any of the following reasons: The following are parameters that require resolution and/or review of clinical history by the Principal Investigator or designee and consultation with the Medical Monitor, prior to continuation of study vaccination(s): If the use of a short tapering course (<2 weeks) of corticosteroids is required, the study vaccinations may be continued after a 4-week washout period, provided that the medical condition requiring this therapy has completely resolved and in the opinion of both the site investigator and Medical Monitor, the continuation of the study vaccinations will not jeopardize the safety of the volunteer.

Follow-Up after Discontinuation of Further Vaccinations
Volunteers who have study vaccinations discontinued due to adverse events will be followed until the adverse event resolves or stabilizes or up to the end of the study, whichever comes last. These volunteers will not be replaced.

Withdrawal from the Study (Early Termination)
Volunteers may be withdrawn from the study permanently for the following reasons: CONFIDENTIAL IAVI Protocol B004, Version 1.0 Page 46 of 79 30Jun11 1. Volunteers may withdraw from the study at any time if they wish, for any reason 2. The Principal Investigator or designee has reason to believe that the volunteer is not complying with the protocol 3. If the Sponsor decides to terminate or suspend the study 4. If a volunteer becomes infected with HIV (through exposure in the community) If a volunteer withdraws or is withdrawn from the study, all termination visit procedures will be performed according to the Schedule of Procedures (Appendices A-B) where possible. Every effort will be made to determine and document the reason for withdrawal.

Data Collection and Record Keeping at the Study Site
Data Collection: All study data will be collected by the clinical study staff using designated source documents and entered onto the appropriate case report forms (CRFs). CRFs will be provided by IAVI and should be handled in accordance with the instructions from IAVI. All study data must be verifiable to the source documentation. A file will be held for each volunteer at the clinic(s) containing all the source documents. Source documentation will be available for review to ensure that the collected data are consistent with the CRFs.
All CRFs and laboratory reports will be reviewed by the clinical team, who will ensure that they are accurate and complete.
Source documents and other supporting documents will be kept in a secure location. Standard GCP practices will be followed to ensure accurate, reliable and consistent data collection.
Source data include but are not limited to: • Signed Informed Consent Documents • Dates of visits including dates of vaccinations • Documentation of any existing conditions or past conditions relevant to eligibility • Reported laboratory results • All adverse events • Concomitant medications • Local and systemic reactogenicity events

Data Entry at the Study Site
The data collected at the study site will be recorded on the CRFs by the study staff. All clinical trial information collected will be entered into a database. To provide for real time assessment of safety, data should be entered into the database as soon as reasonably feasible after a visit occurred.

Data Analysis
The data analysis plan will be developed and agreed upon by the Sponsor, PIs and Collaborating Companies prior to unblinding of the study. The statistician at the Data CONFIDENTIAL IAVI Protocol B004, Version 1.0 Page 47 of 79 30Jun11 Coordinating Centre (DCC), in collaboration with the Principal Investigators, Sponsor, and Collaborating Companies will create tables according to this data analysis plan.
The DCC will conduct the data analysis and will provide interim and final study reports for the Sponsor, Principal Investigator, the SRB and the regulatory authorities, as appropriate.

Sample Size
A total of 75 volunteers (60 vaccine/15 placebo) will be enrolled into 5 groups.

Null Hypothesis
The null hypothesis is based on the primary outcome variables, which are all safety parameters. Let π 0 and π 1 be the proportions of placebo and vaccinated volunteers, respectively, with a primary event. Then the hypothesis is: H 0 : π 0 = π 1 versus H 1 : π 0 < π 1 i.e., a 1-tailed test of whether the proportion of vaccinated volunteers with an event is greater than the proportion of placebo volunteers with an event.

Statistical Power and Analysis
Safety and Tolerability: The rate of local and systemic reactogenicity will be used to assess the differences between vaccine regimens.
The rate of SAEs causally related to the Investigational Product will be used as one measure of the safety of the Investigational Product. AEs that may be temporarily incapacitating (for example, loss or cancellation of work or social activities), which could make an Investigational Product impractical for large scale use if they occur in more than a small proportion of cases, will also be assessed.
All adverse events will be reported, grouped by seriousness, severity and relationship to the Investigational Product (as judged by the investigator).
For any event (e.g., severe AE, SAE related to IP): if none of the volunteers receiving the combination of Investigational Products experiences such an event, then the 2-sided 95% upper confidence limit (CL) for the rate of these events in the population (by the Clopper Pearson method) is 0.06 (n=60) and 0.265 (n=12).
The current sample size will achieve 80% power, at alpha=5%, to detect the following differences in event rates between groups. The calculations are based on Fisher's exact 1-tailed test, using PASS 2008 (www.ncss.com).
IAVI Protocol B004, Version 1.0 Page 48 of 79 30Jun11 For example, if the true rate of events in placebo recipients (n=15) is 5%, then 60 volunteers receiving vaccine will provide 80% power to detect a statistically significant difference of 31% or more (i.e., a rate of ≥36%). The current sample size will achieve 80% power, at alpha=5%, to detect the above differences in event rates between individual or combined groups. The calculations are based on Fisher's exact 2-tailed test, using PASS 2008.
One or more descriptive interim analyses of grouped data may be carried out without unblinding the study to investigators or volunteers. At the end of the study, a full analysis will be prepared according to a pre-specified SAP.

Immunogenicity
Cellular immune responses will be analyzed and compared using binomial methods (Fisher's exact test) to examine for the presence or absence of HIV specific T-cell responses quantified by ELISPOT and ICS. Assays will be performed using the IAVI, HIL, and CRC SOPs and standard reagents for all volunteers.
Presence or absence of antibodies to the vaccine-based HIV gene products will be also analyzed. Assays will be performed in a similar fashion in all volunteers.
A descriptive interim analysis may be performed when all volunteers in a given group have reached one month post boost.

QUALITY CONTROL AND QUALITY ASSURANCE
To ensure the quality and reliability of the data gathered and the ethical conduct of this study, a Study Operations Manual (SOM) will be developed. The SOM includes requirements for reporting and documenting deviations.
IAVI Protocol B004, Version 1.0 Page 49 of 79 30Jun11 Regular monitoring will be performed according to ICH-GCP as indicated in Section 17.3.
An independent audit of the study may be performed by the Sponsor or their designee.
By signing the protocol, the Principal Investigators agree to facilitate study related monitoring, audits, IRB/IEC review and regulatory inspection(s) and direct access to source documents. Such information will be treated as strictly confidential and under no circumstances be made publicly available.

DATA AND BIOLOGICAL MATERIAL
All data and biological material collected through the study shall be managed in accordance with the Clinical Trial Agreement (CTA). Distribution and use of these data will be conducted by agreement of all parties.
The computerized raw data generated will be held by the DCC on behalf of the Sponsor. The CRC will also hold the final data files and tables generated for the purpose of analysis. The Principal Investigator or designees will have access to the clinical database with appropriate blinding.

ADMINISTRATIVE STRUCTURE
The Principal Investigator will be responsible for all aspects of the study at the CRC.

Protocol Safety Review Team
The Protocol Safety Review Team (PSRT) will be formed to monitor the clinical safety data. During the vaccination phase of the trial, the PSRT will review clinical safety data on a weekly basis. An ad hoc PSRT review meeting will occur if any of the members of the PSRT requests a special review to discuss a specific safety issue or as specified in the Study Operations Manual.
The PSRT will consist of the IAVI Medical Monitor(s) and PI or designee from each clinical team. An IAVI Medical Monitor will be the PSRT Chair. Ex officio members will include the IAVI CMO, IAVI Medical Safety Monitor and representatives from Profectus Biosciences Inc. and Ichor Medical Systems, Inc.
Additional PSRT participants may include the following, as needed: • Co-investigators and CRC senior clinical research nursing staff • Laboratory directors • Data management, study statistician and regulatory staff The Terms of Reference for PSRT and procedures to be followed are detailed in the SOM.

Safety Review Board
The Safety Review Board (SRB) will oversee the progress of the study. The SRB will consist of independent clinicians/scientists/statisticians who are not involved in the study. Investigators IAVI Protocol B004, Version 1.0 Page 50 of 79 30Jun11 responsible for the clinical care of volunteers or representative of the Sponsor may not be a member of the SRB.
However, the SRB may invite the Principal Investigator(s) or designee and a Sponsor representative to an open session of the meeting to provide information on study conduct, present data or to respond to questions.
All safety data will be reviewed by an independent Safety Review Board (SRB) once the first 15 volunteers randomized across all groups have reached the M 2.5 and M 6.5 time points.

Content of Interim Safety Review
The SRB will be asked to review the following data: • Summary of reactogenicity (i.e., solicited adverse events) • All clinical adverse events judged by the Principal Investigator or designee to be possibly, probably or definitely related to the Investigational Product • All laboratory adverse events confirmed on retest and judged by the Principal Investigator or designee to be possibly, probably, or definitely related to Investigational Product and/or clinically significant

• All SAEs
An unblinded presentation of all above-noted events will also be available for the SRB for their review if required by any member of the SRB.

Criteria for Pausing the Study
Enrolment and vaccinations will be stopped and a safety review conducted by the SRB for any of the following criteria: • one subject experiences an SAE that is probably or definitely related to the investigational vaccine(s), or two or more subjects experience similar SAEs which are possibly related to the investigational vaccine(s); • there is a subject death assessed as possibly, probably or definitely related to the investigational vaccine(s) • one subject experiences injection site ulceration, sterile abscess or necrosis associated with vaccine administration; or • one subject experiences a severe allergic reaction, such as laryngospasm, bronchospasm or anaphylaxis, associated with vaccine administration.
The Sponsor will request a review by the SRB, or the SRB chair if other SRB members cannot be convened, to be held within 2 business days of the Sponsor learning of the event. The individual volunteer(s)/or study may be unblinded at the discretion of the SRB.
Following this review, the SRB will make a recommendation regarding the continuation or suspension of the vaccinations or the trial and communicate this decision immediately to the Sponsor. The Sponsor then will inform the Principal Investigators without delay.
IAVI Protocol B004, Version 1.0 Page 51 of 79 30Jun11 Additional ad hoc review may be specifically requested by the Sponsor, the Principal Investigator(s) or by the SRB.

Study Supervision
The SRB, the IAVI Chief Medical Officer (CMO) and the IAVI Medical Monitor(s) have access to progress report(s) of this study. Close cooperation will be necessary to track study progress, respond to queries about proper study implementation and management, address issues in a timely manner, and assure consistent documentation, and share information effectively. Rates of accrual, retention, and other parameters relevant to the site's performance will be regularly and closely monitored by the study team, as well as the SRB.

Study Monitoring
On-site monitoring will be conducted to ensure that the study is conducted in compliance with human subjects' protection and other research regulations and guidelines, recorded and reported in accordance with the protocol, is consistent with SOPs, GCP, applicable regulatory requirements and locally accepted practices. The monitor will confirm the quality and accuracy of data at the site by validation of CRFs against the source documents, such as clinical records. The investigators, as well as volunteers through consenting to the study, agree that the monitor may inspect study facilities and source records (e.g., informed consent forms, clinic and laboratory records, other source documents), as well as observe the performance of study procedures. Such information will be treated as strictly confidential and will under no circumstances be made publicly available.
The monitoring will adhere to GCP guidelines. The Principal Investigator will permit inspection of the facilities and all study-related documentation by authorized representatives of IAVI, and Government and Regulatory Authorities responsible for this study.

Investigator's Records
Study records include administrative documentation-including reports and correspondence relating to the study-as well as documentation related to each volunteer screened and/or enrolled in the study-including informed consent forms, case report forms, and all other source documents. The investigator will maintain and store, in a secure manner, complete, accurate, and current study records for a minimum of 2 years after marketing application approval or the study is discontinued and applicable national and local health authorities are notified. IAVI will notify the Principal Investigator of these events.

INDEMNITY
The Sponsor and Institution are responsible to have appropriate liability insurance. For research-related injuries and/or medical problems determined to result from receiving the Investigational Product, treatment including necessary emergency treatment and proper followup care will be made available to the volunteer free of charge at the expense of the Sponsor.

PUBLICATION
A primary manuscript describing safety and immune responses in this trial will be prepared promptly after the data analysis is available. Authors will be representatives of each CRC, the IAVI Protocol B004, Version 1.0 Page 52 of 79 30Jun11 statistical centre, the laboratories and IAVI, subject to the generally accepted criteria of contributions to the design, work, analysis and written report of the study. Manuscripts will be reviewed by representatives of each participating group, as specified in the CTA.

ETHICAL CONSIDERATIONS
The Principal Investigator will ensure that the study is conducted in compliance with the protocol, SOPs in accordance with guidelines laid down by the ICH for GCP in clinical studies, the ethical principles that have their origins in the Declaration of Helsinki and applicable local standards and regulatory requirements. In addition to IEC/IRB and regulatory approvals, all other required approvals will be obtained before recruitment of volunteers, as applicable for individual sites. The Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events ("DAIDS AE Grading Table") is a descriptive terminology that can be utilized for Adverse Event (AE) reporting. A grading (severity) scale is provided for each AE term.
This clarification of the DAIDS Table for Grading the Severity of Adult and Pediatric AE's provides additional explanation of the DAIDS AE Grading Table and clarifies some of the parameters.

I. Instructions and Clarifications
Grading Adult and Pediatric AEs The DAIDS AE Grading Note: In the classification of adverse events, the term "severe" is not the same as "serious." Severity is an indication of the intensity of a specific event (as in mild, moderate, or severe chest pain). The term "serious" relates to a participant/event outcome or action criteria, usually associated with events that pose a threat to a participant's life or functioning.
Estimating Severity Grade for Parameters Not Identified in the Table  In order to grade a clinical AE that is not identified in the DAIDS AE grading table, use the category "Estimating Severity Grade" located on Page 3.

Determining Severity Grade for Parameters "Between Grades"
If the severity of a clinical AE could fall under either one of two grades (e.g., the severity of an AE could be either Grade 2 or Grade 3), select the higher of the two grades for the AE. If a laboratory value that is graded as a multiple of the ULN or LLN falls between two grades, select the higher of the two grades for the AE. For example, Grade 1 is 2.5 x ULN and Grade 2 is 2.6 x ULN for a parameter. If the lab value is 2.53 x ULN (which is between the two grades), the severity of this AE would be Grade 2, the higher of the two grades.
Values Below Grade 1 Any laboratory value that is between either the LLN or ULN and Grade 1 should not be graded.

Determining Severity Grade when Local Laboratory Normal Values Overlap with Grade 1 Ranges
In these situations, the severity grading is based on the ranges in the DAIDS AE Grading  Table. .