A Phase I Double Blind, Placebo-Controlled, Randomized Study of a Multigenic HIV-1 Adenovirus Subtype 35 Vector Vaccine in Healthy Uninfected Adults

Background We conducted a phase I, randomized, double-blind, placebo-controlled trial to assess the safety and immunogenicity of escalating doses of two recombinant replication defective adenovirus serotype 35 (Ad35) vectors containing gag, reverse transcriptase, integrase and nef (Ad35-GRIN) and env (Ad35-ENV), both derived from HIV-1 subtype A isolates. The trial enrolled 56 healthy HIV-uninfected adults. Methods Ad35-GRIN/ENV (Ad35-GRIN and Ad35-ENV mixed in the same vial in equal proportions) or Ad35-GRIN was administered intramuscularly at 0 and 6 months. Participants were randomized to receive either vaccine or placebo (10/4 per group, respectively) within one of four dosage groups: Ad35-GRIN/ENV 2×109 (A), 2×1010 (B), 2×1011 (C), or Ad35-GRIN 1×1010 (D) viral particles. Results No vaccine-related serious adverse event was reported. Reactogenicity events reported were dose-dependent, mostly mild or moderate, some severe in Group C volunteers, all transient and resolving spontaneously. IFN-γ ELISPOT responses to any vaccine antigen were detected in 50, 56, 70 and 90% after the first vaccination, and in 75, 100, 88 and 86% of Groups A–D vaccine recipients after the second vaccination, respectively. The median spot forming cells (SFC) per 106 PBMC to any antigen was 78–139 across Groups A–C and 158–174 in Group D, after each of the vaccinations with a maximum of 2991 SFC. Four to five HIV proteins were commonly recognized across all the groups and over multiple timepoints. CD4+ and CD8+ T-cell responses were polyfunctional. Env antibodies were detected in all Group A–C vaccinees and Gag antibodies in most vaccinees after the second immunization. Ad35 neutralizing titers remained low after the second vaccination. Conclusion/Significance Ad35-GRIN/ENV reactogenicity was dose-related. HIV-specific cellular and humoral responses were seen in the majority of volunteers immunized with Ad35-GRIN/ENV or Ad35-GRIN and increased after the second vaccination. T-cell responses were broad and polyfunctional. Trial Registration ClinicalTrials.gov NCT00851383

• Proportion of volunteers with mild and moderate other adverse events

Secondary:
Immunogenicity: • Proportion of volunteers with HIV-1 specific T-cell responses by ELISPOT assay. If robust responses occur, they will be characterized by multiparameter flow cytometry for detection of intracellular cytokines, functional, surface and memory markers

• Proportion of volunteers showing in vitro inhibition of HIV replication
• Proportion of volunteers with antibodies to HIV antigens • Proportion of volunteers with neutralizing antibodies to Ad35 • Proportion of volunteers with Ad35 vector-specific cellmediated response assessed by ELISPOT assay infected with HIV-1 or HIV-2; who are available for the duration of the trial and willing to undergo HIV testing and use an effective method of contraception; who report low-risk behavior for HIV infection; and who, in the opinion of the principal investigator or designee, understand the study and can provide written informed consent.

STUDY DESIGN
Principal exclusion criteria include: HIV-1 or HIV-2 infection; pregnancy and lactation; a chronic disease which in the opinion of the investigator makes the volunteer unsuitable for the trial; recent Safety and tolerability will be addressed by examining overall rates of reactogenicity events and severe and very severe adverse events and SAEs that might be associated with vaccination and the number of volunteers who experience these events. All clinical and routine laboratory data will be included in the safety analysis. Volunteers will be classified as responders or non-responders based on the results of the immune assays. 8

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, GCP and the applicable regulatory requirement(s).

Instructions:
The Principal Investigator at the study site will sign and date two copies of the protocol signature page indicating that he/she agrees to conduct the study in accordance with the protocol.
One copy of the original, signed protocol signature page will be returned to IAVI where it will be archived. The other copy of the original signed and dated protocol signature page must be filed in the investigator's site file.

INTRODUCTION AND BACKGROUND INFORMATION
According to the Joint United Nations Program on HIV/AIDS and the World Health Organization, as of the end of 2007, 33 million people were estimated to be living with HIV/AIDS, with 96% residing in the developing world It is estimated that in 2007 alone, 2.7 million were newly infected with HIV and 2.1 million died of AIDS 1 .
Sub-Saharan Africa has the largest burden of HIV/AIDS. In sub-Saharan Africa there is a disproportionate impact on females and young people of ages 15-24 years; the ratio of HIVinfected females to males, on average, is 3 to 2, but in the age 15-24 year group the ratio is 3 to 1. HIV prevalence on average is 1.7 times higher in urban areas than in rural areas. HIV prevalence is leveling off, but at an exceptionally high level and with the apparent stabilization in the prevalence attributed to the numbers of newly infected people being roughly the same as the number dying of AIDS-related causes. Worldwide, AIDS is the leading cause of premature death among both men and women aged 15-59 years. Average life expectancy has declined in 38 countries since 1999 primarily as a result of AIDS. In seven African countries where the prevalence exceeds 20%, the average life expectancy of a person born between 1995 and 2000 is 50 years, which is 12 years less than in the absence of AIDS 2 .
Despite considerable progress in care and treatment and prevention efforts, the HIV/AIDS epidemic is still rampant. Beyond the human tragedy of HIV/AIDS, the costs of the epidemic pose a significant impediment to the economic growth and political stability of many countries. In developing countries and in segments of the U.S. population, anti-HIV therapies are frequently beyond financial reach. Accordingly, effective, low-cost tools for HIV prevention, such as a vaccine, are urgently needed to bring the HIV epidemic under control. For this reason, IAVI is committed to the development of safe, effective vaccines to prevent HIV infection and AIDS worldwide.
The effort to develop an effective preventive vaccine against HIV-1 infection is challenged by the wide genetic diversity of HIV-1 among different isolates. Analysis of genomic sequences from different regions in the world has identified at least 9 major subtypes (A, B, C, D, F, G, H, J and K) and dozens of recombinant forms 3 , but together, the A, B and C subtypes represent the viral subtypes responsible for about 75%-85% of new HIV infections in the world 4 5 . Subtype D is present in parts of East Africa. Subtypes B and D are phylogenetically closer to each other than other HIV subtypes 6 .
To be effective, an HIV vaccine will have to induce appropriate immune responses that are potent and long-lasting. Ideally a vaccine would be delivered prior to risk of exposure to HIV. The immune correlates for protection that may be required are not known, but experimental and epidemiological evidence suggests that both high levels of HIV-specific neutralizing antibody and long-lasting CD8+ and CD4+ T-cell responses are needed 7 8 .

Study Rationale
This study is a Phase I dose-escalation clinical trial to evaluate the safety and immunogenicity of Ad35-GRIN/ENV or Ad35-GRIN HIV Vaccines (replicationincompetent recombinant adenovirus serotype 35 expressing HIV-1 subtype A gag, RT, integrase, nef (GRIN) and HIV-1 envelope (ENV)) and administered in a homologous prime-boost regimen by intramuscular route at Months 0 and 6. Version 2.0, 16Oct2009 The recombinant adenovirus vector (rAd) vaccine design is based on the concept of immunization by gene transfer. Recombinant adenovirus vector vaccines offer the positive attributes of immune stimulation by replication incompetent viral vaccines, without adjuvant. The hope is that by using a vaccine that can infect cells and present endogenously produced HIV proteins, such a vaccine could mimic the effectiveness of live attenuated simian AIDS vaccine in macaques. Preclinical studies 9 10 11 12 13 14 and clinical studies 15 16 17 18 19 show that immune responses against HIV can be elicited by direct gene transfer of immunogen-expressing HIV genes via rAd. The major advantage of rAd immunization appears to be its efficacy in transducing host cells and priming the induction of CD8 + cytotoxic T lymphocytes (CTL) responses, which are considered an important element in controlling HIV-1 viral replication 9 20 21 22 23 24 25 26 . There is an additional safety feature in that following entry into the target cells, the HIV-1 gene products will be expressed without the production of infectious adenovirus (Ad) or integration into the host genome. These gene products can be produced in cells that are not actively dividing.
Immunization with more than one immunogen (co-immunization) is an efficient regimen to induce immunity to multiple antigens. HIV-1 envelope (Env) and Gag gene products are the predominant immunogens used in current AIDS vaccines. Few studies however have evaluated possible immune interference when these two antigens are coadministered. Some studies have shown that it is possible to induce immune responses to all proteins of a multi-component vaccine 27 . Other studies have shown loss of immunogenicity against one or more vaccine components 2829 .
Reduced levels of specific antibodies as well as decreased cellular responses have been observed with combinations of genetic immunogens 30 31 . More specifically, immune interference during co-inoculation was studied in mice using env gp160 HIV-1 subtypes A, B and C, DNA gag p37 subtypes A and B, DNA rev subtype B and DNA RT subtype B genes all carried by separate DNA plasmids. The env genes alone induced significantly stronger cellular responses than when env genes were injected together with gag and RT genes 32 .
Mice vaccinated with HIV-1 Env gp120 and Gag p55 plasmids in separate hind legs with each plasmid individually elicited high titer immune responses; however, when plasmids were co-inoculated, there was a reduction in the immune responses elicited to HIV-1 Gag p55 suggesting a possible Env interference 33 . A similar antigen competition, manifested by a relative reduction of CD8+ T-cell responses to Gag and Tat and lymphoproliferation responses to Gag, Env, Tat, and Nef was observed in macaques immunized with combined vaccines 34 .
In humans, DNA plus NYVAC HIV-1 subtype C vaccine regimen induced T cell responses in 90% of vaccinees compared to 33% with NYVAC-HIV-1 vaccine alone. The vaccine-induced T cell responses were however strongest and most frequently directed against Env (91% of vaccines), but lower responses against Gag-Pol-Nef were also observed in 48% of vaccinees 28 . These findings may suggest Env interference. A similar pattern was observed following vaccination with MVA expressing HIV-1 Clade B'/C env, gag, pol, nef, and tat genes. T cell responses were mostly directed to Env, almost none against Gag and a few against other HIV proteins 29 . Version 2.0, 16Oct2009

Experience with Adenoviral Vector Serotype 5 HIV Vaccines
The MRK adenovirus type 5 human immunodeficiency virus type 1 clade B gag/pol/nef vaccine was tested in phase I trial followed by a Phase IIB Test-of-Concept trial jointly with the HIV Vaccine Trial Network. The Vaccine Research Center (VRC) has developed a recombinant serotype 5 adenovector (rAd5) product composed of four rAd5 vectors that encode HIV-1 Env glycoproteins from subtypes A, B, and C, and Gag/Pol polyproteins from subtype B respectively. This vaccine has been evaluated as a single agent in Phase I studies and in Phase I and II studies in healthy human subjects as a boost following vaccination with a plasmid DNA prime vaccine. The study data obtained thus far from the clinical trials with the VRC rAd5 vaccine, as well as from the human clinical trials of rAd5-based vaccines developed by Merck 1335 suggest that rAd5 vaccines are well-tolerated and immunogenic at dosages from 10 9 through 10 11 particle units.
A major potential limitation of rAd5 vector vaccines however, is the high prevalence of pre-existing immunity to adenovirus serotype 5 (Ad5) in human populations, which may diminish vaccine-induced immune responses 9 In addition, interim results of the Phase IIB efficacy study of the Merck rAd5 vaccine (HVTN 502, also known as the STEP Trial) suggest caution in administering rAd5 vector vaccines to subjects with pre-existing Ad5 antibody (Ab) at enrollment 36 .
The STEP Trial enrolled 3000 men and women with an increased risk of exposure to HIV infection. The STEP Trial was halted for futility when the interim data reviewed by the Data and Safety Monitoring Board indicated that the MRK-rAd5 HIV vaccine was not preventing HIV infections and was not reducing the HIV viral load in participants who became HIV-infected. An unexpected safety concern was that there were more HIV infections in male vaccinated participants who already had neutralizing antibody to Ad5 at the time of enrollment (from a prior Ad5 naturally occurring infection) than the male placebo recipients from the same group, particularly among those who were not circumcised. There was only one HIV infection among the women in the STEP Trial, but preliminary data from the halted Phambili trial of the same regimen in South Africa suggest that the pattern in women is similar. Further evaluations of STEP Trial results are ongoing to try to identify the basis for this unexpected observation.
One strategy to overcome the hurdle of pre-existing humoral immunity is to select a human adenovirus serotype with low sero-prevalence, such as Ad35.

Adenoviral Vector Serotype 35 Selection
Many reports have confirmed high rates of Ad5 seroprevalence (with high levels of Ad5 neutralizing antibody), particularly in regions, such as sub-Saharan Africa, which are most at risk for HIV infection and where the need for safe and effective vaccine is most critical 37 38 . Therefore, an active area of research is the development of adenoviral vectors designed to evade dominant Ad5-specific immunity because they are based upon alternative adenovirus serotypes, which are less common.
In adult Africans representative of those who might be enrolled in a Phase I trial, the prevalence of Ad35 neutralizing antibodies was 19% with geometric mean titer (GMT) of 97 (N=360) compared to 89% and GMT at 846 (N=388) for Ad5 (IAVI, unpublished data). In another study, the percentage of Ad35 positive Sub-Version 2.0, 16Oct2009 Sahara African subjects (N=200, 18-65 years) was 17% and GMT 10-fold lower compared to Ad5 (GMT: 60 vs. 600) 29 . In the United States of America, the Ad35 seroprevalence is <7% in adults aged 20-70 years. Serum collected in Europe (Belgium, United Kingdom, and The Netherlands) demonstrated an even lower seroprevalence of 2-7% in healthy volunteers 29 38 .
Thus, serotype Ad35 was selected for development as vaccine vector for the following reasons: (1) In Africa, there is a relatively low prevalence of antibodies to Ad35 (as described above); (2) Pre-existing immunity to Ad5 does not interfere with Ad35 immunogenicity in animals 39 ; (3) Technology is available to IAVI with an improved vector construct back-bone able to generate product at a low virus particle to infectivity (VP:IU) ratio 30 ; (4) Ad35 belongs to adenovirus subtype B that uses highly-expressed CD46 as receptor in contrast to Ad5 belonging to subtype C using the coxsackie-adenovirus receptor (CAR). Ad35 efficiently infects human monocyte-derived immature dendritic cells, which are important targets for eliciting potent and persistent immune responses 30 ; (5) Since Ad35 only weakly interacts with coagulation factor X (FX), it may be preferable to Ad5 whose hexon binds to FX, leading to efficient liver targeting 40 ; (6) Ad35 grows efficiently in HER96 cells; (7) the Ad35 used as vector in this protocol is replication-incompetent.
Outbreaks of acute respiratory disease caused by Ad35 wild type virus are rarely documented in civilian populations. Ad35 wild type is an uncommon serotype associated with very rare cases of serious pulmonary disease, hemorrhagic cystitis, and conjunctivitis, mostly in immuno-compromised patients 41 42 43 44 45 and very rarely in healthy individuals 46 47 48 . More recently, in large cohorts of military recruits and reviews of adenovirus-associated acute respiratory disease in healthy adolescents and adults, Ad35 wild type virus had not been incriminated as a cause of adenovirus illness 49 50 51 .
The recombinant adenoviral serotype 35 vector (rAd35) HIV vaccine in this study, designated Ad35-GRIN/ENV, is designed to test the concept that rAd35 can deliver multiple HIV genes and produce beneficial immune responses with an acceptable safety profile. The gag, RT, IN, nef genes (abbreviated as GRIN) were identified within the HIV-1 sequence, designed as a fusion product, and codon optimized for human cell expression and translation. GRIN was selected based on the evidence that in a worldwide study assessing HIV-1-infected humans, the highest levels of T-cell responses (75-100%) were observed against gag, pol, and nef, regardless of either the donor's origin or the subtype of the infecting virus 52 . ENV gp140 was selected based on data showing that it appears immunogenic as a T-cell based vaccine antigen in humans and is capable of generating unexpectedly significant CTL cross-reactivity to ENV among the different HIV-1 subtypes 53

Primary Objectives
To evaluate the safety of Ad35-GRIN/ENV administered intramuscularly at 0 and 6 months.
To evaluate the safety of Ad35-GRIN administered intramuscularly at 0 and 6 months

Secondary Objectives
To assess the immunogenicity Ad35-GRIN/ENV administered intramuscularly at 0 and 6 months.
To evaluate the immunogenicity of Ad35-GRIN administered intramuscularly at 0 and 6 Months To compare the immunogenicity of Ad35-GRIN administered with and without Ad35-Env.

Other
To study Ad35-GRIN/ENV shedding

Safety:
• Proportion of volunteers with severe and very severe local reactogenicity events (pain, tenderness, erythema, skin discoloration, skin damage (vesiculation, ulceration), induration, formation of crust or scab) • Proportion of volunteers with severe and very severe systemic reactogenicity events (fever, chills, headache, nausea, vomiting, malaise, myalgia, arthralgia) • Proportion of volunteers with severe and very severe other adverse events (including laboratory abnormalities) •

Secondary Endpoints
Immunogenicity: • Proportion of volunteers with HIV-1 specific T-cell responses by ELISPOT assay If robust responses occur, they will be characterized by multiparameter flow cytometry for detection of intracellular cytokines, functional, surface and memory markers • Proportion of volunteers with Ad35 vector-specific cell-mediated response assessed by ELISPOT assay

Study Design
This study is a phase I dose-escalation randomized, placebo-controlled study designed to evaluate the safety and immunogenicity of Ad35-GRIN and Ad35-ENV filled in the same vial and administered as a single, combined vaccine. This is the first administration of this vaccine in humans. The study will be double blind with respect to vaccine or placebo. The vaccine will be administered intramuscularly at months 0 and 6 at three dose levels: 2 x 10 9 , 2 x 10 10 , and 2 x 10 11 vp per dose. Volunteers in Group D will receive Ad35-GRIN at 1 x 10 10 vp administered intramuscularly at months 0 and 6. Volunteers will be randomized to vaccine: placebo in a 10:4 ratio in each group.
The Safety Review Board will authorize the advance to the next dosage level after review of safety data from the first vaccination in the lower dosage group. Prior to enrolment into the mid-dosage group, the SRB will review the clinical and laboratory safety data of the low dosage, based on a compilation of blinded data from the first 9 volunteers enrolled (at least 50% vaccine recipients for a given dosage group). Any additional or subsequent severe or very severe events will be provided to the SRB as an update prior to proceeding with the next dosage level. SRB members will recommend dose-escalation on their medical judgment.
Enrolment into the high dosage group (Group C) will depend on the review of the safety data of the mid-dosage group, following the procedure described above.

Duration of the Study
Volunteers will be screened up to 42 days before vaccination (90 days for Ad35 neutralizing antibody screening) and will be followed for 12 months after the last vaccination (18 months total study participation). It will take approximately 5 months to enroll 56 volunteers. Thus, the total duration of the study would be approximately 23 months.

Study Population
The study population consists of healthy male or female adults aged 18-50 years who are not infected with HIV, do not report risk for HIV infection, available for the duration of the trial, 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 56 volunteers (40 Vaccine recipients, 16 placebo recipients) who meet all eligibility criteria will be included in the study.

Inclusion Criteria
The Investigator will use his/her best clinical judgment in considering a volunteer's overall fitness for trial participation even if all inclusion/exclusion criteria are met.

1.
Healthy males and females, as assessed by a medical history, physical exam, and laboratory tests; 3.
Willing to comply with the requirements of the protocol and available for follow-up for the planned duration of the study (screening plus 18 months, see schedule of procedures);

4.
In the opinion of the Principal Investigator or designee, has understood the information provided. Written informed consent needs to be given before any study-related procedures are performed;

5.
Amenable to HIV risk reduction counseling, committed to maintaining behavior consistent with low risk of HIV exposure through the last required protocol clinic visit, and willing to continue 5 years of annual follow-up contact.

6.
Demonstrates understanding (assessment of understanding will be performed) of the risk for harm observed in the STEP Study results.

7.
Assessed by the clinic staff as being at "low risk" for HIV infection on the basis of sexual behaviors within the 12 months prior to enrolment defined as follows: • Sexually abstinent OR • Had two or fewer mutually monogamous relationships with partners believed to be HIV-uninfected and who did not use illicit drugs ( methamphetamines (crystal meth), heroin, cocaine, including crack cocaine or chronic marijuana abuse)OR • Had two or fewer partners believed to be HIV-uninfected and who did not use illicit 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.
Willing to undergo HIV Testing, HIV counseling and receive HIV Test results;

9.
If sexually active female, using an effective method of contraception (hormonal contraceptive; diaphragm; Intra Uterine Device (IUD); condoms; anatomical sterility in self or partner) from screening until at least 4 months after last vaccination. All female volunteers must be willing to undergo urine pregnancy tests at time points as indicated in the Schedule of Procedures (Appendix A) Version 2.0, 16Oct2009

10.
If sexually active male, willing to use an effective method of contraception (such as condoms, anatomical sterility) from screening until 4 months after the last vaccination. including syphilis, gonorrhoea, non-gonococcal urethritis, Trichomonas vaginalis, symptomatic Herpes genitalis (HSV-2), chlamydia, pelvic inflammatory disease (PID), mucopurulent cervicitis, epididymitis, proctitis, lymphogranuloma venereum, chancroid, or hepatitis B). • Has a high-risk partner either currently or had such a partner within the previous 12 months. 4. Any clinically significant abnormality on history or examination, including history of immunodeficiency or autoimmune disease; use of systemic corticosteroids (the use of topical steroids and inhaled steroids for sinus decongestion are permitted), immunosuppressive, antiviral, anticancer, anti-tuberculosis, or other medications considered significant by the investigator within the previous 6 months; 5. Any clinically significant acute or chronic medical condition that is considered progressive or, in the opinion of the investigator, would make the volunteer unsuitable for the study.

6.
Any of the following abnormal laboratory parameters listed below: 10.
Receipt of blood transfusion or blood products within the previous 6 months; 11. Participation in another clinical study of an investigational product currently, within the previous 3 months or expected participation during this study;

12.
Receipt of another investigational HIV vaccine candidate at any time; 13. History of severe or very severe local or systemic reactogenicity to vaccines or history of severe allergic reactions;

14.
Major psychiatric illness, including any history of schizophrenia or severe psychosis, bipolar disorder requiring therapy, suicidal attempt or ideation in the previous 3 years. 15. Unwilling to forgo donations of blood, sperm, eggs, bone marrow or organs during the study.

16.
Asplenia: any condition resulting in the absence of a spleen

Recruitment of Study Volunteers
Healthy adult male and female volunteers may be recruited through information presented via Internet, in community organizations, hospitals, colleges, other institutions and/or advertisements to the general public. This information will contain contact details and the basic criteria for enrolment in the study.
All methods used for recruitment must be approved by the Ethics Committee prior to implementation. Version 2.0, 16Oct2009 If other recruitment strategies are used, the sponsor needs to be informed. During the recruitment process it is important to ensure full counseling and full informed consent.
Study volunteers satisfying all criteria for enrolment after the screening visit will have to pass an Assessment of Understanding and verbalize understanding of any questions answered incorrectly.

Screening Period
During screening, site personnel will perform the following procedures: • Provide and/or review the Informed Consent Document and answer any questions about the study prior to obtaining written informed consent. • Perform Assessment of Understanding with the volunteer.
• Obtain written informed consent prior to conducting any study procedures. • Initially an IRB approved screening consent form may be used to allow for early screening of subjects. • The results from this screening may be used to determine eligibility for this protocol as long as the tests and information are within the time period specified in the eligibility section.
If the volunteer agrees to participate, site personnel will: • Provide a screening questionnaire to the volunteer for completion • Perform an HIV risk assessment • Perform a complete medical history (including concomitant medication) • Perform a general physical examination, including height, weight, vital signs (pulse, respiratory rate, blood pressure and temperature), examination of skin, respiratory, cardiovascular and abdominal systems, and an assessment of cervical and axillary lymph nodes. • Conduct pre-HIV test counseling • Collect blood and urine specimens for all tests, as indicated in the Schedule of Procedures (Appendix A). Perform a pregnancy test for all female volunteers.
Screening laboratory test(s) may be repeated once at the discretion of the principal investigator or designee to investigate any isolated abnormalities.
Volunteers will be screened first for Ad35-specific serum neutralizing antibodies, within 90 days prior to the date of first vaccination. All other screening procedures must occur within 42 days prior to the date of first vaccination. If screening procedures are not performed within these time periods, they must be repeated. The complete medical history may be replaced by an interim medical Version 2.0, 16Oct2009 history and the Volunteer Information Sheet should be reviewed with the volunteer.
If a volunteer has signed the informed consent form, but does not meet the eligibility criteria, the records must be kept at the site.

VACCINATION VISIT
Prior to vaccination (Months 0 and 6), site personnel will: • Review the Informed Consent Document with volunteers.
• Fill in eligibility checklist and decide the eligibility of the volunteer to participate in the vaccine trial. • Perform an HIV risk assessment • Answer any questions about the study • Review interim medical history (including concomitant medications) • Review (screening) safety laboratory data of previous visit.
• Perform a directed physical examination, including vital signs (pulse, respiratory rate, blood pressure and temperature), examination of vaccination site as well as an assessment of axillary lymph nodes and any further examination indicated by history or observation). • Conduct pre HIV-test counseling.
• Collect blood, urine specimens as well as oropharyngeal swabs for all tests including viral shedding study, as indicated in the Schedule of Procedures (Appendix A). The volunteer will be assigned a unique allocation number according to the instructions specified in the Study Operations Manual.
The Investigational Product will be administered as specified in Section 8.4, Administration.
Site personnel will closely observe volunteers for at least 30 minutes after vaccination for any acute reactogenicity events. At the end of the observation period site personnel will: • Record vital signs (pulse, respiratory rate, blood pressure and temperature) • Assess any local and systemic reactogenicity • Assess any other adverse events.
• Provide the volunteer with a Memory Card and instructions to assist in the collection of reactogenicity events and AEs following vaccination For subsequent vaccination visits (the 'preferred' window for the 2 nd vaccination is +/-7 days and the 'allowable' window is +/-14 days) site personnel will perform the same procedures as above with the following exceptions: • Review the routine safety laboratory parameters (Section 9.1.5) 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 12.1) • Conduct pre HIV-test counseling if an HIV Test is required (Appendix A) or provide post-test counseling if the results of a prior HIV test are being provided to the volunteer.
A volunteer will be considered as ENROLLED once she/he has been randomly allocated to a specific vaccination regimen.

Post-Vaccination Visits
The volunteer will be asked to return to the clinic on Day 3 (+/-1 day) and on Day 7 (+/-2 days) and Day 14 (+/-2 days) after each vaccination for an assessment.
The study personnel will review the Memory card with the volunteer and record the information in the clinic chart.
The following procedures will be conducted at this visit: • Review of interim medical history and use of concomitant medications.
• If symptoms are present, perform a symptom-directed physical examination. • Assess local and systemic reactogenicity, as well as any other adverse events. • Collection of blood and urine specimens, as well as oropharyngeal swabs for all tests including viral shedding study, as indicated in the Schedule of Procedures (Appendix A).
In case of adverse event(s), the volunteer will be assessed and followed up by the clinical team. Supplemental visit(s) for further investigation can be planned at the discretion of the clinical and Principal Investigators. Supplemental visit(s) may be recommended if clinically indicated or to clarify observations. All the required supportive care will be provided and referral services will be facilitated.

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

Unscheduled Visits/Contact
Unscheduled Visits/contacts are visits/contacts that are not described in the Schedule of Procedures (Appendix A). They may be performed at any time during the study. Unscheduled visits may occur: Version 2.0, 16Oct2009 • 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.
• In the event that a volunteer presents to the study site after having missed a scheduled study visit outside a scheduled visit window. • For other reasons, as requested by the volunteer or site investigator.
All unscheduled visits will be documented in the volunteers' study records and on applicable source documents.

Final Visit/Early Termination Visit
Assessments and procedures will be performed according to the Schedule of Procedures (Appendix A).
Site personnel will: • Review any adverse events and concomitant medications • Perform a general physical examination, including height, weight, vital signs (pulse, respiratory rate, blood pressure and temperature), examination of skin, respiratory, cardiovascular and abdominal systems, and an assessment of cervical and axillary lymph nodes.

Informed Consent Process
A sample informed consent document is provided by the sponsor to the site. The site specific Informed Consent Document will be submitted and approved by IAVI and then the Institutional Ethics Committees of the site before use.
Volunteer Information Sheet A qualified authorized member of the study staff will obtain informed consent by reviewing the Volunteer Information Sheet with the volunteer.
The following study specific elements are included: 1. It is unknown whether or not the Investigational Product will protect against HIV infection or disease or might enhance susceptibility to infection or disease 2. It may be possible that the vaccinated volunteer will develop antibodies against HIV following vaccination, which may produce a positive result in a routine HIV Antibody Test. Provisions have been made to distinguish between response to vaccine and HIV infection during and after the study. In case the volunteer has a positive result in a routine HIV Antibody Test, he/she will be followed until the result is no longer positive. Version 2.0, 16Oct2009 3. It is imperative that each volunteer should avoid any risky behavior for HIV infection during the entire period of the trial. 4. A sexually active volunteer should use a reliable form of contraception from screening, during the vaccination period until 4 months after the last injection. 5. A placebo will be administered to some volunteers in this study and these volunteers will receive placebo throughout the study.

Informed 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 ask questions.
The volunteer's consent to participate must be obtained by him/her signing and dating the informed consent form witnessed by a member of the study team. The members of the site personnel who are involved in conducting the informed consent discussions must also sign and date the Informed Consent Form.
The signed/marked and dated informed consent document must remain at the study site. A copy of the signed and dated informed consent form will be offered to the volunteer to take home if the volunteer is willing to receive the consent form. 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 counseling regarding a volunteer's participation in the study ONLY with the written consent of the participating volunteer.

Medical History and Physical Examination
At screening, a comprehensive medical history will be collected, including details of any known previous reaction to vaccinations, history of sexually transmitted diseases, contraceptive practices, and history of epilepsy.
A general physical examination includes the following: height, weight, vital signs (pulse, respiratory rate, blood pressure and temperature), examination of skin, respiratory, cardiovascular and abdominal systems, and an assessment of cervical and axillary lymph nodes. This examination will be conducted at screening and termination visits.
At each other study visit, an interim medical history and symptom directed physical examination will be performed. A directed physical examination will include vital signs, examination of vaccination site and any further examination indicated by history or observation. Version 2.0, 16Oct2009

HIV Risk Assessment, HIV Testing and HIV Test Counseling
Study staff will assess volunteers for past and current risk of HIV infection. A screening questionnaire and other tools may be used, according to site-specific procedures.
Additionally, study staff will perform pre-HIV test counseling (prior to collecting blood for an HIV test) and post-HIV test counseling (when HIV test results are available) according to the Schedule of Procedures (Appendix A). For more information on HIV testing and HIV-test counseling, see Section 10.

Family Planning Counseling
Study staff will counsel volunteers about the importance of preventing pregnancies and use of condoms, as well as other effective family planning methods. Condoms may be provided and volunteers may be referred to a family planning clinic if a contraceptive prescription is required, according to standard practice of the study site.
The family planning counseling will be performed at time points according to the Schedule of Procedures (Appendix A).

Blood Collection
Up to 20 mL of blood will be collected at the Screening Visit and up to 85 mL of blood will be collected at later visits, usually from the antecubital fossa, according to the Schedule of Procedures (Appendix A).
All specimens will be handled according to the procedures specified in the Study Operations Manual, as specified by the Core Lab SOPs and Study Analytical Plan.
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.

Compensation for Participation
Volunteers will be compensated 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 consent form or the site-specific Volunteer Information Sheet approved by the site Ethics Committee.

Randomization and Blinding
Volunteers will be identified by a unique volunteer identification number.
The randomization schedule will be prepared by the statisticians at the Data Coordinating Center (DCC) prior to the start of the study. Volunteers will be assigned a specific allocation number. An unblinding list will be provided to the Pharmacist by the DCC for emergency use only.
This study is double-blinded. Study staff (investigator and clinical personnel monitoring the safety and laboratory assay results) and volunteers will be blinded with respect to the allocation of Investigational Product or Placebo.
Blinding will not apply to the dosage group assignment (A, B, C or D).
An unblinded pharmacist will prepare the syringe and deliver it to the person who injects the investigational product. Since vaccine and placebo may differ in their physical appearance, the person administering the dose should not be the same person who performs the assessment of safety and reactogenicity.
Volunteers will be informed about their assignment (vaccine or placebo) at the end of the study when all data are collected and all queries are resolved. If the study volunteer is unblinded during the course of the study and becomes aware of treatment assignment, 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.

Unblinding Procedure for Individual Volunteers
Unblinding of an individual volunteer may be indicated in the event of a medical emergency for which the clinical management/medical treatment of the volunteer would be altered by knowledge of the group assignment. Whenever feasible, the IAVI Medical Monitor should be contacted prior to unblinding.
The unblinded information should be restricted only to a small group of individuals involved in clinical management/medical treatment of the volunteer (e.g., treating physician) and the blind should be maintained for those responsible for the study assessments.
The reasons for unblinding should be documented and the Data Coordinating Centre should be notified. The procedures and contact numbers for unblinding are outlined in the Study Operations Manual.

Description
The Ad35-GRIN/ENV is supplied as a frozen sterile formulation in a 2-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 9 vp or 2x10 10 vp or 2x10 11 vp per dose. Ad35-GRIN vials contain 0.725 mL of vaccine. The volume of administration, 0.5 mL, will deliver a final dosage of 1x10 10 vp. 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 150mm 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.

Placebo
Placebo is provided as a frozen sterile suspension in a 2 mL vial with a butyl rubber stopper and aluminum seal. Each vial contains 0.725 mL of placebo. The volume of administration is 0.5 mL. The final formulation buffer (see description above) will be used as a placebo. The placebo will be manufactured, filter sterilized and filled under GMP in the same fill-finish container as will be used for the vaccine. Placebo is colorless.
The summary of the Investigational Product is shown in Table 2.

Shipment and Storage
Authorization to ship the Investigational Product to the site will be provided in writing by the sponsor, upon confirmation that all required critical documents for shipment authorization are completed. The Investigational Product will be shipped to the site according to the required storage conditions.
Ad35-GRIN/ENV and placebo are stored at -70°C or below. The different formulations of the Ad35 GRIN/ENV and Ad35-GRIN vaccines and placebo are identified by unique lot numbers. Additionally, all the vials from all the three dosage levels and placebo have a date of manufacturing, storage temperature, dose volume, the name of the manufacturer and the US cautionary statement.

Dispensing and Handling
The Investigational Product will be dispensed as specified in the Study Operations Manual.
Each vial containing placebo or vaccine should be thawed at ambient temperature in the pharmacy. Thawed vials to be gently swirled to mix the contents and aspirated into a syringe as soon as possible. In case of an unplanned delay, keep the vial at 2-8 o C. Vaccine should be utilized as soon as possible. The vaccine should be used within 3 hours post thawing. Draw 0.5 mL of the vaccine into the syringe, label the syringe and transfer to the pharmacist for vaccine administration without delay.

Administration
Investigational Product will be administered according to the Schedule of Procedures (Appendix A).
The preferred site of first administration is the deltoid muscle of the non-dominant upper arm (for example, injection in the left arm if the volunteer uses mainly the right arm) unless contraindicated for another reason when receiving a single injection of Ad35-GRIN/ENV or Ad35-GRIN or placebo. The booster injection will be injected in the same arm.
Further information on the administration of the Investigational Product is supplied in the Study Operations Manual.

Accountability and Disposal
All used vials will be returned to the Investigational Product dispenser or pharmacy at the end of each vaccination visit. The date, vial allocation number and location of storage of the returned vials will be recorded.
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 Standard Operating Procedures.

Safety Assessments
Data on local and systemic reactogenicity will be collected by structured interview, using specific questions. Data on other adverse events will be collected with an open-ended question. All data will be recorded on the appropriate source documents.

Local reactogenicity
The presence of local reactogenicity will be assessed at the time points specified in the Schedule of Procedures (Appendix A).
Local reactogenicity (pain, tenderness, erythema/skin discoloration, skin damage [vesiculation/ulceration], induration, formation of crust or scab) will be assessed and graded using the Appendix B, 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 (Appendix A).
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.
Fever, chills, headache, nausea, vomiting, malaise, myalgia and arthralgia will be assessed and graded using the Appendix B, Adverse Event Severity Assessment Table, as a guideline.

Other adverse events
Occurrence of other adverse events (including Serious Adverse Events) will be collected following an open question to volunteers at the time points indicated in the Schedule of Procedures (Appendix A). The adverse events will be graded using the Appendix B, Adverse Event Severity Assessment Table, as a guideline.
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.
Concomitant receipt of Investigational Products, including other HIV vaccines is prohibited during the study.
If clinically indicated, non-live vaccines (non-HIV) or live attenuated influenza vaccine may be given up to 14 days before study vaccination(s) or after post-vaccination blood draw (i.e., 2 weeks after study vaccinations).
Live-attenuated vaccines (non-HIV) may be given 60 days before study vaccination(s) or after the post-vaccination blood draw. However, the study vaccination(s) should not be given if there are any continuing symptoms from recently administered non-HIV vaccines. In this situation, the Principal Investigator should consult with the IAVI Medical Monitor before administering the next study vaccination.
The administration of immunoglobulin will be followed by the discontinuation of vaccinations. In this situation, the Principal Investigator should consult with the IAVI Medical Monitor before administering the next study vaccination.
If the use of a short tapering (< 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 the IAVI 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 3 shows the laboratory parameters that will be measured routinely. These parameters will include hematology, clinical chemistry, immunological assays and urinalysis. The samples for these tests will be collected at the time points indicated in the Schedule of Procedures (Appendix A). 9.1.6 Specific screening tests: Volunteers will be screened to exclude the following diseases:

Routine laboratory parameters
• Hepatitis B: positive for hepatitis B surface antigen (HbsAg) • Hepatitis C: positive for hepatitis C antibodies (HCV antibodies) • Syphilis: confirmed diagnosis of active syphilis (RPR & TPHA or equivalent)

Antibody Responses
• Antibodies against HIV proteins will be measured at times indicated on the Schedule of Procedures (Appendix A).
• Anti-Ad35 vector neutralizing antibodies will be assessed at times indicated in the Schedule of Procedures (Appendix A).

Cellular Responses
Immunogenicity assays, including ELISPOT for monitoring the number of circulating T-cells that can be stimulated to produce cytokines, will be performed at time points indicated in the Schedule of Procedure (Appendix A, using peptide pools representing all or a portion of the encoded antigen(s). Further characterization of phenotype and functional properties of responding T-cells will be performed using multi-parameter flow cytometry and/or the Virus Inhibition Assay (ability of PBMC to restrict the growth of HIV in vitro). An algorithm may be applied to determine which time points are analyzed.
Further studies may be carried out using a.) Peptide pools designed to determine the specific epitopes recognized and b.) Peptides from different HIV-subtypes and c.) Peptides from Ad35 vector proteins. Selected T-cell responses may be further characterized for HLA restriction and additional markers on the responding cells, such as markers for activation or homing to mucosal tissues.
At each time point indicated in the Schedule of Procedures (Appendix A), using the procedure provided by the IAVI Core Laboratory, vials of frozen peripheral blood mononuclear cells (PBMC) each containing approximately 10 7 PBMC will be taken for immunogenicity analysis (ELISPOT, CFC) and/or quality control assays at the IAVI Core Laboratory. These samples will be shipped promptly, according to an agreed upon schedule, included in the Study Analytical Plan. Version 2.0, 16Oct2009

PBMC, Serum and Plasma Storage
Samples of cryo-preserved PBMC, plasma and serum will be taken at time points indicated in the Schedule of Procedures (Appendix A) for purposes of standardization, quality control and for future assays related to HIV vaccine research and development. These samples will be archived and only a code will identify the samples.
For the PBMC processing and potentially some immunogenicity assessments, the laboratory personnel will be trained as necessary by the sponsor and provided with a written procedure manual.
The samples described in Sections 9.2.2 and 9.2.3 will be shipped routinely from the site to the IAVI Core Laboratory. The majority of the immunological testing will be performed at the IAVI Core Laboratory in accordance with IAVI standard operating procedures and standard reagents.

HLA Typing
Samples for HLA typing will be collected at the time point indicated in the Schedule of Procedures (Appendix A).
HLA typing will be performed on samples for volunteers vaccinated at each dosage level, provided that T-cell responses are detected at that dosage level.

HIV test
Samples will be tested at the time points indicated in the Schedule of Procedures (Appendix A). 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 the time points indicated in the Schedule of Procedures (Appendix A).
The results of the pregnancy test must be negative prior to vaccination.

Antibody response to the Ad35 vector
Since pre-existing immunity to Ad35, as well as antibodies induced by Ad35 vector itself, may impair subsequent immune responses to the proteins of interest expressed by the vector, antibodies to the vector will be assessed at time points specified in the Schedule of Procedures (Appendix A). Samples for anti-Ad35 neutralizing antibodies will be sent to the Core Laboratory or another suitable laboratory for testing.

Viral inhibition assay
Viral inhibition assays will be carried out at IAVI Core Laboratory at time points indicated in the Schedule of Procedures (Appendix A).

Viral shedding
Oropharyngeal swabs and urine specimens will be collected in 9 volunteers in each dose group at time points indicated in the Schedule of Procedures (Appendix A), basically day 0, day 14 post 1st injection and prior to booster injection at month 6. The samples will be frozen and adenovirus culture will be performed if Ad35-specific DNA PCR is found positive. In addition, specimens for adenovirus investigation will be collected as clinically indicated according to the medical judgment of the investigator within the first 14 days post immunization for any reported respiratory or genito-urinary tract or diarrheal illness or conjunctivitis, unless another cause is revealed by the diagnostic investigations (e.g. bacterial UTI, streptococcal pharyngitis, positive test for influenza antigen). .

Definition
An adverse event (AE) is any untoward medical occurrence in a volunteer administered Investigational Product; an AE does not necessarily have a causal relationship with the Investigational Product. An AE can therefore be any unfavorable 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 is ultimately the responsibility of the principal investigator.
The following general criteria should be used in assessing adverse events as mild, moderate or severe at the time of evaluation: • Mild: Mild discomfort; Minimal or no limitation of daily activities; Medical intervention not required; • Moderate: Moderate discomfort; Some limitation of daily activities but able to work part-time or full-time with some assistance; May require minimal or no medical intervention; • Severe: Severe discomfort; Marked limitation of daily activities, unable to work; Requires medical intervention; • Very severe: Symptoms causing inability to perform basic self-care functions OR Medical or operative intervention indicated to prevent permanent impairment, persistent disability, or death Guidelines for assessing the severity of specific adverse events and laboratory abnormalities are listed in Appendix B, Adverse Event Severity Assessment Table.

Relationship to Investigational Product
The relationship of an (S)AE is assessed and determined by the Principal Investigator or designee. All medically indicated and available diagnostic methods (e.g., lab, 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 Investigational Product and/or other cause.
The following should be considered for the assessment of relationship of adverse events to the investigational Product:

• 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 assessment should be reported as one of the following: Not Related: clearly explained by another cause (concurrent disease, concomitant medication, environmental or toxic factors, etc.).
Unlikely: more likely explained by another cause (concurrent disease, concomitant medication, environmental or toxic factors, etc.).
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 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 ICH 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 study staff becoming aware of the event. All SAEs should be sent by fax to a designated fax number or e-mailed to MAreport@iavi.org according to SAE Reporting Guidelines (see Study Operations Manual).
To discuss Investigational Product related SAEs or any urgent medical questions related to the SAE, the site investigator should contact the IAVI medical monitor 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), relationship assessment to the investigational product by the investigator.
The Principal Investigator or designee is required to write a detailed written report with follow up until resolution or until the medical condition 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 the FDA, the Safety Review Board and other study sites where the same Investigational Product is being tested.
More details on SAE definitions and reporting requirements are provided in the SAE Reporting Guidelines (see Study Operations Manual).

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 the harm caused by the Investigational Product or study procedures, this treatment will be provided free of charge.
If a volunteer has an adverse event and/or abnormal laboratory value that is known at the time of study vaccination(s), the specifications of Section 12.1 will be followed.
Volunteers will be followed until the adverse event resolves or stabilizes or up to the end of the study, whichever comes last. If at the end of the study, an adverse event (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 adverse event, 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 case report forms. However, serious complications of pregnancy that meet SAE criteria specified in the Section 10.4 of this Protocol (e.g., eclampsia, spontaneous abortion, etc.) should be reported as SAEs. For follow up on a pregnancy, refer to Section 12.2.
If a female volunteer becomes pregnant during the study, vaccinations will be discontinued and the volunteer followed until the end of pregnancy. Approximately 2-4 weeks after delivery, the baby will be examined by a physician to assess his/her health status and the results will be reported to IAVI.

Intercurrent HIV Infection
HIV infection cannot be caused by the Investigational Product. If a volunteer is found to be HIV-infected, study vaccinations must be discontinued and the volunteer followed according to procedures described in Section 12.2.
Intercurrent HIV infection in study volunteers, although not considered an SAE, must be reported promptly to IAVI using the designated case report forms. IAVI will report intercurrent HIV infections to the FDA using the same procedure as SAE reports. However, serious medical conditions associated with the HIV infection that meet SAE criteria specified in the section 10.4 of this Protocol (e.g. sepsis, PCP pneumonia, etc.) should be reported as SAEs using SAE Report Form.

HIV Testing
Only volunteers who are not HIV-infected at screening will be enrolled into the study. Version 2.0, 16Oct2009 Volunteers will be tested for HIV-1 and HIV-2 antibodies as indicated in the Schedule of Procedures (Appendix A) or as needed, if medical or social circumstances arise.
If the routine post-vaccination HIV Antibody test is positive, a predetermined algorithm will be followed to distinguish an immune response to the vaccine from an HIV infection through exposure in the community.
If a volunteer is found to be HIV-infected, a newly drawn blood specimen will be collected for confirmation.
Volunteers who have a positive HIV-antibody test(s) as a result of vaccineinduced HIV antibodies, rather than a true HIV infection (false positive HIV test), will have their test result reported as "Not infected with HIV-1 or HIV-2" (to prevent unblinding of volunteer and staff) and will be followed up until the test becomes negative. At the end of the study, these volunteers will be offered a continuing follow-up until the test becomes negative.
Should a volunteer require an HIV test outside the study for personal reasons, it is recommended that the volunteer contact the site personnel first. The HIV test can be drawn at the clinical site and then processed at the independent laboratory as above. Written evidence of HIV status (HIV-infected or HIVuninfected) will be provided upon request.
All volunteers will receive HIV prevention counseling and pre-HIV-test and post-HIV-test counseling as specified in Section 11.3.1 Counseling.

Social Discrimination as a Result of an Antibody Response to Vaccine
The aim is 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, according to site procedures.

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

Counseling
The volunteer will be counseled by the study counselors. The counseling process will assist the volunteer with the following issues: • Psychological and social implications of HIV infection • Whom to inform and what to say • Implications for sexual partners • Implications for child-bearing Version 2.0, 16Oct2009 • Avoidance of transmission to others in future

Referral for Support and/or Care
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.
If a volunteer becomes HIV-infected during the study, he/she will be referred for medical care.
HIV-infected pregnant women will be referred for prenatal care and to a program for the Prevention of Mother to Child Transmission (PMTCT).
The pregnant volunteer will be followed according to timeline as specified in Section 10.6

Discontinuation of Vaccinations
The Principal Investigator as well as the IAVI Medical Monitor will discuss the circumstances relating to any volunteer discontinuing further vaccinations or being considered for discontinuation or deferring of vaccinations. Volunteers will be discontinued from further vaccination for any of the following reasons: A severe local reaction involving the major part of the injected arm circumference. 8.
Life-threatening adverse event following study vaccinations, unless not related to the Investigational Product and fully resolved. 10.
Any immediate hypersensitivity reaction judged to be definitely related to the Investigational Product.
Participating in another clinical study of an Investigational Product.

Follow-up after Discontinuation of Further Vaccinations
Volunteers in whom study vaccinations are 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.
Follow-up of HIV-infected individuals who have received Investigational Product will be determined by the Principal Investigator and the IAVI Medical Monitor.
Follow-up of pregnant volunteers will be done as specified in Section 10.6.

Withdrawal from the Study (Early Termination)
Volunteers may be withdrawn from the study permanently for the following reasons: 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.

Follow-up Withdrawal from the Study (Early Termination)
If the volunteer withdraws from the study, all termination visit procedures will be performed according to the Schedule of Procedures (Appendix A) if possible. Version 2.0, 16Oct2009 Every effort will be made to determine and document the reason for withdrawal from the study.

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 documentation. 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 and remain separate from volunteer identification information (name, address, etc.) to ensure confidentiality.
Standard GCP practices will be followed to ensure accurate, reliable and consistent data collection.

Source documents include but are not limited to:
• Signed Informed Consent Documents

Data Collection and Transfer at the IAVI Core Laboratory
Data generated at the IAVI Core laboratory will be transferred directly to the Data Coordinating Centre.

Data Entry at the Study Site
The data collected at the site will be entered onto the electronic CRFs by the designated study staff. To provide for real time assessment of safety, data should be entered as soon as reasonably feasible following a study visit. Version 2.0, 16Oct2009

STATISTICAL CONSIDERATIONS
The statistician at the Data Coordinating Centre (EMMES Corporation), in collaboration with the sponsor and the principal investigator (or designee), will create tables according to a data analysis plan that has been reviewed and agreed to by the principal investigator (or designee). The EMMES Corporation will conduct the data analysis and will provide interim and final study reports for the principal investigator (or designee), the sponsor and the regulatory authorities, as appropriate. Prior to an analysis, additional monitoring visits will take place if necessary to validate the data held on the database, as well as all consent forms and dispensing records. Data files will be prepared by EMMES from a 'frozen' dataset for that particular analysis.

Sample Size
A total of 56 volunteers (40 Vaccine/16 placebo) will be entered into each of the 4 groups (10 vaccine/ 4 placebo recipients in each group) scheduled to receive Ad35-GRIN/ENV or Ad35-GRIN vaccines or placebo. All injections will be intramuscular (IM). An over-enrolment of about 10% (1-2 volunteers per arm) will be permitted to facilitate prompt enrolment.

Statistical Power and Analysis
Safety will be assessed by analyses of the following primary end-points (events), where the unit of analysis in each case will be the proportion of volunteers with at least one event: a) Severe and very severe local reactogenicity events b) Severe and very severe systemic reactogenicity events c) Severe unsolicited adverse events, including severe and very severe laboratory abnormalities d) Severe unsolicited adverse events that are possibly, probably or definitely related to vaccine or placebo, including any severe and very severe laboratory abnormalities e) Serious adverse events f) Mild or moderate local or systemic reactogenicity events, or adverse events The rate of Serious Adverse Events related to the Investigational Product will be used as one measure of the safety of the Investigational Product. Adverse Events 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 as to whether or not they qualify as SAEs, their severity assessment, and their relationship to the Investigational Product (as judged by the investigator).
Cellular immune responses will be analyzed using binomial methods to examine for the presence or absence of HIV specific T-cell responses quantified by Version 2.0, 16Oct2009 ELISPOT and cytokine flow cytometry (CFC). Assays will be performed using the IAVI Core Laboratory SOPs and standard reagents for all volunteers.
Presence or absence of antibodies to HIV proteins will be also analyzed. Assays will be performed in a similar fashion in all volunteers.
Ad35-specific neutralizing antibodies will be analyzed post vaccination (percentage of volunteers positive and mean titer).
Based on the previous experience with IAVI Phase I Investigational Product studies, it is expected that the amount of missing, unused or spurious data will be insignificant. Unused and spurious data will be listed separately and excluded from the statistical analysis. Missing data will be excluded from the statistical analysis.

Prior to enrolment of the mid-dose group:
Prior to enrolment into the mid-dose group, the SRB will review the safety of the low dose, based on a compilation of blinded data from the first 9 volunteers enrolled. Any additional subsequent severe or very severe events will be provided to the SRB as an update prior to proceeding with the next dosage level. Based on the binomial distribution, if none of the 9 volunteers experiences an event, then the upper 95% confidence limit (CL) for the rate of these adverse events in the population is 33.6%. Similarly, if one or two events are observed then the corresponding upper 95% CLs are 48.2% and 60.0%, respectively.

Prior to enrolment of the high-dose group:
Enrolment into the high dose group (Groups C) will depend on the review of the safety data of the mid-dose group, following the procedure described above.

Vaccine versus Placebo within a dose group
For comparison of active vaccine (N=10) versus placebo (N=4) within the same dosage group, there will be 80% power to detect a statistically significant (p<0.05) difference of 76% if the event rate in the placebo group is 1% to 5%, based on Fisher's exact 1-tailed test. This power will apply to a comparison of the anti-GRIN responses in Ad35-GRIN and AD35-GRIN/ENV high dose group.

Overall Vaccine versus Placebo
For comparison of active vaccine (N=40) versus placebo (N=16), there will be 80% power to detect a statistically significant (p<0.05) difference of 28.5% and 32.7% if the event rate in the placebo group is 1% or 5%, respectively, based on Fisher's exact 1-tailed test.

Comparison of Active Vaccine groups
With 10 volunteers receiving active vaccine in each dose group, if the rate of events in one group is 5%, 10%, 20% or 30%, then there will be 80% power to detect statistically significant (p<0.05) differences of about 65%, 67%, 68% and 65%, respectively, based on Fisher's exact 2-tailed test.

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 has been developed. Regular monitoring will be performed according to ICH-GCP as indicated in Section 17. 3.
An independent audit of the study may be performed, if appropriate, at the discretion of the sponsor.
By signing the protocol, the Principal Investigator, agrees 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 all biological material collected throughout the clinical trial shall be the joint property of the investigators and IAVI and managed in accordance with the Clinical Trial Agreement. Distribution and use of these data will be conducted by agreement of both parties.
The computerized raw data generated will be held by the DCC on behalf of the sponsor. The study site 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 study database with appropriate blinding.

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

Safety Review Board
The SRB will review blinded safety data and make recommendations regarding the dose-escalation. The SRB will also be convened to consider any significant safety issue which arises during the study. The SRB will consist of independent clinicians/scientists/statisticians who are not involved in the study. Investigators responsible for the clinical care of volunteers or representative of the sponsor may not be members of the SRB.
However, the SRB may invite the Principal Investigator 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. Version 2.0, 16Oct2009 The review of study data by the SRB will take place at a pre-determined interval or may be specifically requested (see Section 17.1.2 Indications for Discontinuation of Vaccinations in all Volunteers).

Content of Interim Review
The SRB will be asked to review the following data: • All moderate, severe and very severe clinical adverse events/reactogenicity judged by the Principal Investigator or designee to be possibly, probably or definitely related to the Investigational Product, or • All moderate, severe and very severe laboratory adverse events confirmed on retest and judged by the Principal Investigator or designee to be possibly, probably, or definitely related to Investigational Product.
• All Serious Adverse Events, independent of relationship to the Investigational Product.
• All available safety data prior to the administration of the 6-month booster dose.
The SRB will then recommend to the Principal Investigator and the Sponsor whether or not to escalate the dose.

Indications for Discontinuation of Vaccinations in all Volunteers
If 3 or more of the volunteers participating in this study develop an SAE judged definitely, probably or possibly related to the Investigational Product, the Principal Investigator or designee and the sponsor will request a review by the SRB. The study will be suspended pending a review of all safety data by the SRB. The study may be unblinded at the discretion of the SRB.
Following this review, the SRB will make a recommendation to the Sponsor and the Principal Investigator regarding the continuation of the study.

Study Supervision
The Principal Investigator, the IAVI Chief Medical Officer, the Medical Monitor and the Senior Clinical Program Manager will be provided 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 effective information sharing. 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 and other research regulations and guidelines, recorded and reported in accordance with the protocol, is consistent with locally accepted HIV counseling practices, standard operating procedures, Good Clinical Practice (GCP) and applicable regulatory requirements.
The monitor will confirm the quality and accuracy of data at the site by validation against the source documents, such as clinical records, and against the database when applicable. The Investigators and volunteers, by giving consent, 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 Good Clinical Practice 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 relevant to 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 for 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 follow-up 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, based on the data compiled by the IAVI statistical centre. Authors will be representatives of the trial site, the statistical centre, the laboratories and IAVI, subject to the generally accepted criteria of contributions to the design, work, analysis and writing of the study. Manuscripts will be reviewed by representatives of each participating group as specified in the Clinical Trial Agreement.

URINALYSIS Standard International Units are listed in italics
Hematuria (microscopic) 6 -10 RBC/HPF > 10 RBC/HPF Gross, with or without clots OR with RBC casts Transfusion indicated