Immunogenicity of Fractional Doses of Tetravalent A/C/Y/W135 Meningococcal Polysaccharide Vaccine: Results from a Randomized Non-Inferiority Controlled Trial in Uganda

Background Neisseria meningitidis serogroup A is the main causative pathogen of meningitis epidemics in sub-Saharan Africa. In recent years, serogroup W135 has also been the cause of epidemics. Mass vaccination campaigns with polysaccharide vaccines are key elements in controlling these epidemics. Facing global vaccine shortage, we explored the use of fractional doses of a licensed A/C/Y/W135 polysaccharide meningococcal vaccine. Methods and Findings We conducted a randomized, non-inferiority trial in 750 healthy volunteers 2–19 years old in Mbarara, Uganda, to compare the immune response of the full dose of the vaccine versus fractional doses (1/5 or 1/10). Safety and tolerability data were collected for all subjects during the 4 weeks following the injection. Pre- and post-vaccination sera were analyzed by measuring serum bactericidal activity (SBA) with baby rabbit complement. A responder was defined as a subject with a ≥4-fold increase in SBA against a target strain from each serogroup and SBA titer ≥128. For serogroup W135, 94% and 97% of the vaccinees in the 1/5- and 1/10-dose arms, respectively, were responders, versus 94% in the full-dose arm; for serogroup A, 92% and 88% were responders, respectively, versus 95%. Non-inferiority was demonstrated between the full dose and both fractional doses in SBA seroresponse against serogroups W135 and Y, in total population analysis. Non-inferiority was shown between the full and 1/5 doses for serogroup A in the population non-immune prior to vaccination. Non-inferiority was not shown for any of the fractionate doses for serogroup C. Safety and tolerability data were favourable, as observed in other studies. Conclusions While the advent of conjugate A vaccine is anticipated to largely contribute to control serogroup A outbreaks in Africa, the scale-up of its production will not cover the entire “Meningitis Belt” target population for at least the next 3 to 5 years. In view of the current shortage of meningococcal vaccines for Africa, the use of 1/5 fractional doses should be considered as an alternative in mass vaccination campaigns. Trial Registration ClinicalTrials.gov NCT00271479


Confidential Background
Although meningococcal meningitis can occur anywhere in the world, the largest epidemics occur in Africa, in an area known as the "meningitis belt". This belt includes 18 sub-Saharan countries from The Gambia, in the west, to Ethiopia, in the east. Until this year, the majority of outbreaks in that region has been caused by Neisseria meningitidis serogroup A along with a smaller contribution of serogroup C 1 . Major epidemics have occurred in the meningitis belt every 5 to 10 years since the beginning of the 20 th century 2 . The cycles of outbreaks have, however, been less obvious in the last decades, hence making it more difficult to predict their occurrence. In 1996, the largest meningococcal epidemic so far affected the meningitis belt, resulting in 200,000 reported cases and 20,000 deaths (WHO figures, substantially underestimated) 3 .
N. meningitidis serogroup W135 (W135) was first described in 1968. Cases caused by this capsular polysaccharide type have since been reported sporadically worldwide. This serogroup was, until now, not contributing significantly to epidemics and therefore was considered of little epidemiological importance. A limited number of N. meningitidis serogroup W135 cases have been confirmed in Africa since the early 80's 4,5 . In 1994, Kwara and coll. reported cases of meningitis caused by serogroup W135 in Mali (during a serogroup A epidemic) and in 1995, such strains were found in The Gambia 6 . Major concerns arose when a W135 strain was identified from cases in a large outbreak among Hajj pilgrims coming back from Mecca, Saudi Arabia, in 2000 7 . Cases of W135 meningococcal disease were subsequently reported in Europe 8,9 , the United States 10 and from African countries in the meningitis belt 11 . The fear of a worldwide spread of this strain was confirmed when an increased number of W135 cases were  13 . The high case fatality ratio in cases infected with N. meningitidis serogroup W135 disease compared to other serogroups have raised concern internationally 9 . 7 Confidential Rational Epidemiological surveillance and epidemic incidence thresholds have been used for early detection of meningococcal meningitis epidemics. Most outbreaks so far have been caused by either N. meningitidis serogroup A or N. meningitidis serogroup C. In outbreak situations, the WHO recommends treating meningitis cases with a single dose of intramuscular oily chloramphenicol 14,15 and initiating a reactive mass vaccination campaigns with the meningococcal A+C polysaccharide vaccine for the entire population from 2 to 30 years of age.
Questions have been raised about the current strategy of mass vaccination and its costeffectiveness, and debates are still ongoing [16][17][18] . On middle term/long term perspective, several pharmaceutical industries are developing a meningococcal tetravalent conjugate vaccine A/C/Y/W135 19 . However, this tetravalent conjugate vaccine is not expected to be on the market before 5 to 10 years and will probably be even less affordable for developing countries. Only a meningococcal serogroup C conjugate vaccine is currently on the market for £17.95 per dose 20 . The Meningitis Vaccine Project (MVP), a partnership between the Program for Appropriate Technology in Health (PATH) and the WHO is currently exploring possibilities to develop a meningococcal conjugate vaccine against serogroup A. This monovalent conjugate A vaccine is expected to be available for the [2007][2008] epidemic season at an affordable price for developing countries 21 .
The current dose used in the licensed tetravalent A/C/Y/W135 polysaccharide vaccine is 50µg of each polysaccharide component. During the 1980's, researchers from the Walter Reed Army Institute of Research (WRAIR) did extensive works on the immunogenicity of meningococcal polysaccharide vaccines in adults. A first study performed by Griffiss, Brandt and coll. reported that doses of 5 µg of group Y and group W 135 polysaccharides were as effective as doses of 50µg in inducing production of bactericidal antibody amounts correlating with functional immunity 22 . A second study concluded that doses of 7.5µg (Y and W) and 15µg (A and C) were sufficient to induce equivalent binding and bactericidal antibody responses as 50µg 23 . Similar conclusion was reported in a third trial 24 .
In a more recent study from Granoff et al., 1/50 (1 mcg) of the ordinary dose of tetravalent A/C/Y/W135 vaccine was given 25 . The antibody responses to A and C have been measured, and this low dose was sufficient to mount a C response in most of the subjects, but the dose was less effective in eliciting a response to A. The antibody responses to W135 and Y were not reported.
This approach already has a successful precedent as shown by the work of O. Levine and his colleagues with an Hib conjugate vaccine in Central America: Similar functional antibody activities was elicited using one-half or one-third dose of the vaccine 26-28 . 9 Confidential Hypothesis Lower doses of each A/C/Y/W135 component of the meningococcal polysaccharide vaccine could confer a similar functional immunogenic response as the dose of 50µg currently being used, and subsequently be equally protective.
It would potentially bring two major benefits. Firstly, it would increase the number of tetravalent vaccine doses available on the market. Secondly, it would decrease the cost of the individual vaccine dose (see economic analysis "Appendix 1"). As a result, more people could be vaccinated, and thereby protected against the disease, and to a lower price.
Results obtained with the study on the tetravalent A/C/Y/W135 polysaccharide vaccine would be valid for the trivalent A/C/W135 polysaccharide vaccine.

Main objective
To evaluate the use of reduced dose tetravalent meningococcal polysaccharide vaccine to control outbreak caused by N. meningitidis serogroup W135

Primary objectives
• To measure the immunogenicity of a dose corresponding to one fifth of the amount of the licensed meningococcal A/C/Y/W135 polysaccharide vaccine, i.e. 10µg for each component • To measure the immunogenicity of a dose corresponding to one tenth of the licensed meningococcal A/C/Y/W135 polysaccharide vaccine, i.e. 5µg for each component

Secondary objectives
• To determine the pharyngeal carriage of N. meningitidis and in particular W135 strains in the study population.
• To determine the natural immunity towards N. meningitidis serogroup A, C, Y and W135 before immunisation in the study population.
• To measure a possible waning of immunity at one year and at two years after immunisation.
• To measure the immune response after challenging with a second dose of the commercialised meningococcal A/C/Y/W135 polysaccharide vaccine after one year, in a group of volunteers who have received a reduced dose in day 0.
• To create a network of institutions (NIPH, Ugandan MOH, Mbarara University, WHO, Epicentre, MSF) able to co-ordinate efforts and to give a more appropriate response for later outbreaks.
• To strengthen local research capacities through the training of local researchers and technicians.

Investigation plan
Epicentre is the promoter of this clinical trial and will assure the co-ordination of the study with the partners.

Study area and study population
The study must be conducted in a population in Africa that has not been exposed to outbreaks of W135, but is a population at risk of meningococcal meningitis epidemics. Mbarara District was chosen, principally because this area has not experienced a recent epidemic of meningococcal meningitis, a factor that excludes the presence of a high level of background antibodies from previous infections. Furthermore, neighbouring countries, such as Burundi and Rwanda, have faced in 2002 and 2003 respectively outbreaks of meningococcal meningitis serogroup A forcing health authorities to vaccinate massively at risk population 29,30 . By its geographical situation, possible outbreaks of the same etiological pathogen is a permanent threat for Uganda, and more specifically for the Mbarara region. Mbarara is also a research base for the epidemiology agency Epicentre in Uganda. Thus it provides highly competent human resources and logistic capacity essential for such a study.
Within the district of Mbarara, Rwampara County was chosen as the site for recruitment of study participants. Stability of the study population was assessed before choosing the site of immunization. The district directorate of health services for Mbarara District provided advise in the selection of this site. Rwampara County is located 15 km southwest of Mbarara Town, on the Mbarara-Kabale Highway. The county is mostly rural with scattered trading centers and population in Rwampara is predominantly subsistence farmers.
The main health unit in the county or health sub district is the Kinoni Health Center IV. There is a medical officer in charge of the health centre and a team of nurses and support staff assists him.
The health sub district is also a suitable site for this interventional study because it has had long standing collaboration with Mbarara University, department of Community Health. The area is participating in mosquito and child health projects both coordinated by Mbarara University.
There is also a community based health care (CBHC) program where lay persons are trained as facilitators of health activities in the community. This infrastructure will be utilized in the follow up of study volunteers.

Study design
The study design is a randomised single-blind controlled trial.

Vaccines
The vaccine doses to be tested are based on the reports of Mc Griffiss et al.

First injection
Three groups will be used in this clinical trial: • Group 1 will receive a dose of 50µg of each component of A/C/Y/W135 polysaccharide vaccine (currently used dose = 0.5 ml) • Group 2 will receive a 1/5 volume (0.1 ml) of the tetravalent vaccine (10µg of each component) • Group 3 will receive a 1/10 volume (0.05 ml) of the tetravalent vaccine (5µg of each component of meningococcal A/C/Y/W 135 polysaccharide vaccine) Volunteers will receive randomly one of the 3 doses of vaccine.

Second injection: Challenge with a second dose of the commercialised meningococcal
Widespread use of meningococcal A and C polysaccharide vaccines has raised concerns about inductions of hypo-responsiveness to these polysaccharides 31-33 . In order to assess this parameter, a random cluster of 40 volunteers from the group 1, 2 and 3 will be given a second injection with 50µg of each component of A/C/Y/W135 polysaccharide vaccine one year after the first injection.

Injection of the vaccines
The vaccines use for the study are manufactured by Aventis Pasteur Inc., commercialised as Menomune®.
The vaccine will be injected subcutaneously.
Low volume syringes will be used to inject lower doses of vaccine.
-0.80 ml for the 50µg dose -0.16 ml for the 10µg dose -0.08 ml for the 5µg dose (Luer syringe B/BRAUN Omnifix-F®) These dosages are based on the availability for the study of monodose A/C/Y/W135 polysaccharide vaccine currently on the market.

Recruitment of the participants
Stability of the study population will be assessed before choosing the site of immunization.
In collaboration with the Mbarara health authorities and the Mbarara University Teaching Hospital, the recruitment of the participants for the clinical trial will be done on a volunteer basis.
Volunteers will be recruited in the age group from 2 to 20 years old. The age distribution of the volunteers will be matched to the Ugandan age distribution of the 2-20 years old extracted from the "2002/03 Uganda National Household Survey" (see Appendix 3).
Before inclusion, a parental agreement as well as an informed signed consent, are mandatory for all participants (see regulatory and ethical considerations).
An interview will be performed for each volunteer (children, parents or guardians) included in the study. The questionnaire will collect medical history information and vaccination history.
Confidential information (e.g name, contact home address) will be separately recorded and kept disjointedly from the Case report form, in order to respect privacy and confidentiality for participants.

Randomization
Once the informed consent signed and clinical examination done, each participant will be randomly allocated to one of the 6 trial groups using a bloc randomization method stratified by The study is a single-blind randomized controlled clinical trial. After randomization, participants will receive one of the 3 doses of vaccine described previously. The injection will be made without the participant knowing which dosage of vaccine he/she will receive. In addition, the immunogenicity response assay carried out at NIPH will be performed blinded, that is without the laboratory knowing the group to which the participants belong until completion of the analyses.

Sample size in each group
The study is a non inferiority trial to prove that the reduced doses of the vaccine elicit equivalence responses to the currently licensed vaccine dose.
According to the literature, individual responses to all four polysaccharide antigens as determined by serum bactericidal assays (SBA) [defined as fourfold or greater increase between pre and postsera antibody after immunisation] is expected to be over 90% 34-37 . These studies have been performed in healthy adult volunteers in USA.
In the study we plan to conduct in Uganda, presenting a population of young children and potential subclinical nutrition deficiencies among the volunteers, the antibody responses to immunisation with the meningococcal A/C/Y/W135 polysaccharide vaccine is expected to be lower. We have used an estimate of 80% for the calculation of the sample size. Assuming a higher proportion of responders will lead to lower sample sizes but will be less realistic.
The needed sample size in this study has been calculated by choosing a significance level (one sided) of α of 5% and power of 80%. We expect equal proportions of responders (above a cut off on the protective antibody levels) in all groups given the vaccine being 80%. We have decided to accept a difference limit ∆ of 10%. This gives a required sample size of 198 persons in each group. Because the reference group (license vaccine) will be used for 2 comparisons, a correction of (N= n s comparison of no ) was applied (Lellouch & Lazar, 1974), bringing that group to 280. The calculations have been performed with nQuery Advisor® software.
Taking into account the problems relative to the interpretation of non inferiority studies, particular attention will be given to ensure a good quality follow up of all volunteers. We plan to recruit 280 volunteers in the reference group and 220 in the two other groups -to allow for loses to follow up -a total of 720 participants.

Eligibility criteria
• Inclusion criteria -Volunteers should not be suffering of severe chronic disease or a known congenital or acquired immunodeficiency. A medical exam will be performed by a medical doctor before inclusion.
-Volunteers must be living in Mbarara district and within 15 Km from the site of immunization. Volunteers should be residents of the chosen site and should express no plan of moving from this area during the study period.
-Volunteers must be available for follow-up for the duration of the study (minimum of 24 months).
-Able and willing to provide information so that the participant may be located. • Exclusion criteria -Volunteers with severe chronic disease or with a general condition requiring hospital admission.
-Volunteers with a known congenital or acquired immunodeficiency (e.g. HIV). Diagnosis will be presumptive based on the medical background and the clinical examination. No serological HIV testing will be performed.
-Evidence of any concomitant infection at the time of presentation (including rashes other than scabies, ear, nose or throat infections, and abnormal respiratory system examination).
-The patient has any other underlying disease that compromise the diagnosis and the evaluation of the response to the study medication.
-History of serious adverse reactions to vaccines such as anaphylaxis or related symptoms such as hives, respiratory difficulty, angioedema and abdominal pain.

Confidential
-Malnutrition: The nutritional assessment of children aged 24-59 months, a weight-forheight (W/H) index will be calculated. This index is expressed in standard deviations of a normalised distribution of a reference population 38 (National Centre for Health Statistics, USA). Children under 5 years old with a Z-score inferior to -2 will be excluded. For children over 5 or adults, the clinical examination will be considered.
-Pregnant women and lactating women are not eligible for this trial. All women of childbearing age must provide a urine sample for pregnancy testing before inclusion and, for sub-group "b", before the second vaccine injection. NOTE : For the subgroup "b", if a women of child-bearing age becomes pregnant during the first year of follow-up, she will not receive a second injection and will be excluded from the second year analysis.

Adverse Events (AE) and Serious Adverse Events (SAE)
The investigator is responsible for the detection and documentation of events meeting the criteria and definition of an AE or SAE as provided in this protocol. Clinical officers identified for the study, will be under the responsibility of the investigator and be in charge of clinical examination and the assessment and follow-up of AE and SAE.

Definition of an AE
Any untoward medical occurrence in a patient or clinical investigation subject, temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product.
An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the use of a medicinal product. For marketed medicinal products, this also includes failure to produce expected benefits (i.e. lack of efficacy), abuse or misuse.

Examples of an AE includes:
• Pain at the site of injection Examples of an AE does not include a/an: • Medical or surgical procedure (e.g., endoscopy, appendectomy); the condition that leads to the procedure is an AE. • Situations where an untoward medical occurrence did not occur (social and/or convenience admission to a hospital).
• Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present or detected at the start of the study that do not worsen.

Definition of a SAE
A serious adverse event is any untoward medical occurrence that, at any dose:

Time Period, Frequency, and Method of Detecting AEs and SAEs
AEs and SAEs will be collected by Medical Officers or Clinical Officers, from the time of informed consent to the time the patient completes the study (day 30 after injection, or withdraw).
At Day 0, participants will be observed for at least 1 hour after the injection (similar surveillance will be implemented for the second injection at 12 months for subgroups "b").
From thereon, assessment of AEs and SAEs will be done on a weekly based consultation in the month following the day of the vaccine injection. Subjects will be ask to return to the immunization site with tracers hired for volunteers not presenting for follow-up.
Apart from scheduled visits for follow-up, participants can come to the Epicentre clinic located at Mbarara University Teaching Hospital for clinical examination whenever necessary.
Adverse events or reactions not previously documented in the study will be recorded in the adverse experience section of the patient's case record form (CRF). The nature of each experience, date and time (where appropriate) of onset, duration, severity and relationship to he injection should be established.
Adverse events or reactions already documented in the CRF i.e. at a previous assessment and designated as 'continuing' should be reviewed. If these have resolved, the documentation in the CRF should be completed.
NB. If an adverse experience changes in frequency or severity during a study period, a new record of the experience will be started.
Ask the patients a non-leading question such as: "Have you appeared or felt different in any way since starting the new treatment / since the last assessment?"

Recording of AEs and SAEs
When an AE/SAE occurs, it is the responsibility of the investigator to review all documentation (e.g., hospital progress notes, laboratory, and diagnostics reports) relative to the event. The investigator will then record all relevant information regarding an AE/SAE on the CRF.
The investigator will attempt to establish a diagnosis of the event based on signs, symptoms, and/or other clinical information. In such cases, the diagnosis should be documented as the AE/SAE and not the individual signs/symptoms.

Assessment of Intensity of AEs and SAEs
The investigator will make an assessment of intensity for each AE and SAE reported during the study. The assessment will be based on the investigator's clinical judgement. The intensity of each AE and SAE recorded in the CRF should be assigned to one of the following categories: • Mild: An event that is easily tolerated by the subject, causing minimal discomfort and not interfering with everyday activities. • Moderate: An event that is sufficiently discomforting to interfere with normal everyday activities.
• Severe: An event that prevents normal everyday activities.
An AE that is assessed as severe should not be confused with a SAE. Severity is a category utilised for rating the intensity of an event; and both AEs and SAEs can be assessed as severe.

Assessment of Causality of AEs and SAEs
The investigator is obligated to assess the relationship between investigational product and the occurrence of each AE/SAE. The investigator will use clinical judgement to determine the relationship. Alternative causes, such as natural history of the underlying diseases, concomitant therapy, other risk factors, and the temporal relationship of the event to the investigational product will be considered and investigated. The investigator will also consult the CIB/IB and/or Product Information, for marketed products, in the determination of his/her assessment.
The investigator will provide the assessment of causality as per instructions on the SAE form in the CRF.

Follow-up of AEs and SAEs
After the initial AE/SAE report, the investigator is required to proactively follow each subject and provide further information on the subject's condition.
All AEs and SAEs documented at a previous visit/contact and are designated as ongoing, will be reviewed at subsequent visits/contacts. All AEs and SAEs will be followed until resolution, until the condition stabilises, until the event is otherwise explained, or until the subject is lost to follow-up. Once resolved, the appropriate AE/SAE CRF page(s) will be updated. The investigator will ensure that follow-up includes any supplemental investigations as may be indicated to elucidate the nature and/or causality of the AE or SAE. This may include additional laboratory tests or investigations, histopathological examinations, or consultation with other specific health care professionals.

Serology
Serum samples will be collected from each volunteer at the time of vaccination and at 4 weeks, 12 months (cluster group of 40 persons in each group 1,2 and 3), 13 months (cluster group of 40 persons in each group 1,2 and 3), and 24 months after vaccination. We allow a delay of more or less 10 days with regard to the planned date of blood collection.
Ten ml of whole blood will be collected for antibody determinations.
The immune responses to the different doses of the A/C/Y/W135 vaccine will be assayed with enzyme linked immunosorbent assay (ELISA) and serum bactericidal activity (SBA). In ELISA IgG antibodies to each separate polysaccharide A, C, Y and W135 will be measured by a standard method 39,40 . In addition, in a subset of sera (10% of the samples) will be tested using an ELISA method measuring higher-avidity antibodies 41 . The SBA assays for each serogroup will be based on the NIPH experience with measuring SBA against serogroup B 42,43 and modified according to Borrow et al. 44 . The SBA assay will be performed with the tilt method (reaction mixture after incubation is plated onto agar plates to count surviving colony forming units (CFUs)) applying baby rabbit complement 44,45 . When establishing the SBA, human complement will also be used in a subset of sera (10% of the samples) for comparison 46,47 . The importance of complement source will initially be tested with both human and rabbit complement in pre and post vaccination sera (4 weeks) from 25% of the vaccinees from group 1a ( = 60) sera from individuals who have got 50 mcg (covering all age groups). Depending of the results from this analysis, we may eventually come back and study other groups with human complement.
Standardised inocula for each serogroup of organism examined will be used. A four-fold increase in the SBA assay titer is chosen as a criterion for significant increase in bactericidal antibodies. In addition, geometric mean titers will also be calculated. As methods for serogroups W 135 and Y in particular are not validated, modifications of the methods may be used in addition to the primary methods. The ELISA and SBA assays will be performed at NIPH. Emphasis will be put on analysing the response to W 135 first.
Total 720 720 720 720 720 120 120 120 120 720 720 NB: Taking into account the public health priorities and the logistic constraints, ELISA and SBA testing will be performed on serogroup A and W135 in order to allow publications of the results of ELISA 1&2 and SBA 1&2 in the six months following the beginning of the clinical trial.
ELISA and SBA testing of the serogroup C and Y will be performed later. Sera for ELISA 3 and SBA 3 will be collected the same day before the second injection in the subgroups b. Sera for ELISA 4 and SBA 4 will be collected 4 weeks after the second injection in the sub-groups b.

Quality control
To provide confirmatory testing as a quality control, a collaboration has been established with Manchester Public Health Laboratory, in the UK. This laboratory will analyse about 10% of the samples from day zero and day 4 weeks.

Confidential
Carriage study Posterior pharyngeal samples will be collected from the volunteers at 0 and 4 weeks. Samples will be plated directly on selective (VCN) chocolate agar and forwarded immediately to the laboratory in the country where the study is performed. Plates will be incubated for 2 days.
Meningococci will be identified by standard laboratory methods 48 . One colony from each throat culture will be subcultured twice and preserved frozen for further analyses. The serogroup will be determined by agglutination with commercial antisera. Preserved isolates will be forwarded to the NIPH, Oslo for further characterization using monoclonal antibodies and molecular techniques.
Volunteers who will be found carriers of N. meningitidis of a homologous serogroup at any time will be excluded from the analysis of response to that polysaccharide.

Statistical analysis
The definition of a adequate responder is any participant with a four-fold increase in the SBA assay titer.
The baseline characteristics of the participants of each experimental arm will be presented using descriptive statistics and compared by Chi2 or Student's t-test analyses.
Results of the carriage study will be presented using descriptive statistics. Numbers of carriers of each homologous serogoup of N. meningitis will be presented.
The proportion of adequate responses in each group will be expressed as a percentage with associated 95% confidence intervals (response to vaccine rate). Results will be primarily based on a per-protocol analysis, however an intention-to-treat (ITT) analysis will also be performed.
Results for vaccine efficacy will be based on SBA and used to test for non-inferiority.
Safety and tolerability analysis will be conducted on all patients who received at least one vaccine injection. Adverse Events will be listed in order of frequency, and described according to median duration, severity, and likelihood that they were related to the study vaccine. The incidence of Adverse Events in the three groups will be estimated. Any Severe Adverse Events will be described in detail. Outcomes of Adverse Events and Serious Adverse Events (recovery, sequelae, etc.) will also be presented.

Study duration
The total duration of the study is 36 months.
-First analysis: 6 months Results of the carriage study and the antibody determinations (serology at 0 and 4 weeks) are expected to be available after 6 months. Taking into account the public health priorities and the logistic constraints, ELISA and SBA testing will be performed on serogroup A and W135 in order to allow publications of the results of ELISA 1 & 2 and SBA 1 & 2 in the six months following the second blood sample (ELISA 2 & SBA 2). ELISA and SBA testing of the serogroup C and Y will be performed later.
-Second analysis: 18 months Results the antibody determinations (serology at 12 months and 13 months) are expected to be available after 18 months.
-Third analysis: Results the antibody determinations (serology at 24 months) are expected to be available after 30 months.
-Intermediate analysis: An intermediate analysis will be performed at the end of the phase 1 study period. At this stage, if the hypothesis tested appears not to be valid, the phase 2 and 3 study will be aborted, after approval of the scientific committee.

Regulatory and ethical considerations
Regulatory Authority Approval Epicentre, NIPH will obtain approval to conduct the study from the Mbarara University of

Confidential
The protocol must also receive official backup from the Epidemiology and Surveillance department of the Uganda Ministry of Health.
The protocol will also be presented to the National Committee for Medical Research Ethics in Norway. The same standards apply for the conduct of this study in the hosting country as for an equivalent study in Norway. The study will start only when all the ethical approvals have been obtained.

Ethical Conduct of the Study and Ethics Approval
This study will be conducted in accordance with "good clinical practice" (GCP) and all applicable regulatory requirements, including, where applicable, the 1996 version of the Declaration of Helsinki (Appendix 2).
The investigator (or sponsor, where applicable) is responsible for ensuring that this protocol, the site's informed consent form, and any other information that will be presented to potential subjects (e.g., advertisements or information that supports or supplements the informed consent) are reviewed and approved by the appropriate IEC/IRB. The investigator agrees to allow the IEC/IRB direct access to all relevant documents. The IEC/IRB must be constituted in accordance with all applicable regulatory requirements.
If the protocol, the informed consent form, or any other information that the IEC/IRB has approved for presentation to potential subjects is amended during the study, the investigator is responsible for ensuring the IEC/IRB reviews and approves, where applicable, these amended documents. The investigator must follow all applicable regulatory requirements pertaining to the use of an amended informed consent form including obtaining IEC/IRB approval of the amended form before new subjects consent to take part in the study using this version of the form.

Expected benefits, risks and inconveniences
Uganda, more specifically the South-West region including Mbarara district, is at risk of meningitis outbreaks. Indeed, neighbouring countries have recently experienced epidemics of meningococcal meningitis. By participating in the study, subjects will be protected against all 4 serogroups of N. meningitides if such an outbreak occurs.
The study vaccine, Menomune ® -A/C/Y/W-135 , is commercialised for more than 20 years in the United States, Canada and Europe. Adverse events are well documented and rare (see Appendix 3). Risks and inconveniences for the participants should be minor in the study. Nevertheless, precautions will be taken by the investigators for the safe and effective use of the vaccine (e.g.

Confidential
Epinephrine injection will be immediately available to treat unexpected anaphylactic or other allergic reactions).
Incentive to the study Incentive will systematically be proposed to all participants of the study: impregnated mosquito nets and transport refund will be provided when necessary.

Data Monitoring and Safety Committee
A Data Monitoring and Safety Committee will be created, including a staff member of the immunization department of the WHO and a person from the Epidemiology and Surveillance department of the Ugandan Ministry of Health. The Committee will be in charge of verifying the quality of the databases used for analysis. If a Meningococcal meningitis epidemic occurs during the study, measures based on the international recommendations will be taken in collaboration with the Ministry of Health for prevention and case treatment 49 . In this case, the Committee will reserve itself the right to evaluate the need for a "rescue" immunization for participants who have received fractional doses of the A/C/Y/W135 vaccine according to the period of the outbreak, the point in time of the study and the age group to be vaccinated.

Informed consent
Informed consent will be obtained before the subject can participate in the study. The contents and process of obtaining informed consent will be in accordance with all applicable regulatory requirements.
Information should be given in both oral and written form, in presence of an eye witness, whenever possible and deemed appropriate by the ERC/IRB. Only subjects giving their authorisation and signing an informed consent will be enrolled. For all participants, parents or guardians will be approached for consent.
Consent forms will be translated into the local language, Runyankore, and must use a vocabulary fully comprehensible to the prospective patient, their relatives, guardians or, if necessary, legal representatives. Informed consent shall be documented by the use of a written consent form approved by the ERC/IRB and signed by the patient or the patient's legally authorised representative.

Confidential
The written consent document will embody the elements of informed consent as described in the Declaration of Helsinki and will also comply with local regulations. The explanation should include the aim of the study, the expected benefits for the participants, the risks and inconveniences.
Consent must be documented either by the dated signature of the patient or of an independent witness. The signature confirms the consent is based on information that has been read and understood. If the volunteer is unable to write their signature then a thumbprint may be used.
If the volunteer is unable to read the information herself, full and comprehensive information must be communicated to the potential in the presence of a witness. The witness will be an independent third party i.e. not a nominated co-investigator. The witness will sign the informed consent form (testifying that informed consent has been given verbally) along with the investigator (or her nominated representative).
Each patient's signed informed consent form must be kept on file by the investigator for possible inspection by Regulatory Authorities.

Confidential
These estimates of the cost of using fractional dose of trivalent vaccine (results 1) at 1/5 or 1/10 show clear reductions of cost by person vaccinated: respectively, 40% and 32% than the expected cost with the new trivalent vaccine. The cost per person vaccinated for 1/5 or 1/10 of the dose is at the same level as the bivalent A+C polysaccharide vaccine.
The benefit of reduced dose will be even higher, 28% and 19% than the expected cost with the tetravalent vaccine (results 2).
The calculations from the MSF intervention in an emergency context may have underestimated the real benefit of using a fractional dose.

INTRODUCTION
It is the mission of the physician to safeguard the health of the people. His or her knowledge and conscience are dedicated to the fulfilment of this mission.
The Declaration of Geneva of the World Medical Association binds the physician with the words, "The health of my patient will be my first consideration", and the International Code of Medical Ethics declares that, "A physician shall act only in the patient's interest when providing medical care which might have the effect of weakening the physical and mental condition of the patient." The purpose of biomedical research involving human subjects must be to improve diagnostic, therapeutic and prophylactic procedures and the understanding of the aetiology and pathogenesis of disease.
In current medical practice most diagnostic, therapeutic or prophylactic procedures involve hazards. This applies especially to biomedical research.
Medical progress is based on research which ultimately must rest in part on experimentation involving human subjects.
In the field of biomedical research a fundamental distinction must be recognized between medical research in which the aim is essentially diagnostic or therapeutic for a patient, and medical research, the essential object of which is purely scientific and without implying direct diagnostic or therapeutic value to the person subjected to the research.
Special caution must be exercised in the conduct of research which may affect the environment, and the welfare of animals used for research must be respected.
Because it is essential that the results of laboratory experiments be applied to human beings to further scientific knowledge and to help suffering humanity, the World Medical Association has prepared the following recommendations as a guide to every physician in biomedical research involving human subjects. They should be kept under review in the future. It must be stressed that the standards as drafted are only a guide to physicians all over the world. Physicians are not relieved from criminal, civil and ethical responsibilities under the laws of their own countries.

Confidential
1. Biomedical research involving human subjects must conform to generally accepted scientific principles and should be based on adequately performed laboratory and animal experimentation and on a thorough knowledge of the scientific literature.
2. The design and performance of each experimental procedure involving human subjects should be clearly formulated in an experimental protocol which should be transmitted for consideration, comment and guidance to a specially appointed committee independent of the investigator and the sponsor provided that this independent committee is in conformity with the laws and regulations of the country in which the research experiment is performed.
3. Biomedical research involving human subjects should be conducted only by scientifically qualified persons and under the supervision of a clinically competent medical person. The responsibility for the human subject must always rest with a medically qualified person and never rest on the subject of the research, even though the subject has given his or her consent.
4. Biomedical research involving human subjects cannot legitimately be carried out unless the importance of the objective is in proportion to the inherent risk to the subject.
5. Every biomedical research project involving human subjects should be preceded with careful assessment of predictable risks in comparison with foreseeable benefits to the subject or to others. Concern for the interests of the subject must always prevail over the interests of science and society.
6. The right of the research subject to safeguard his or her integrity must always be respected. Every precaution should be taken to respect the privacy of the subject and to minimize the impact of the study on the subject's physical and mental integrity and on the personality of the subject.
7. Physicians should abstain from engaging in research projects involving human subjects unless they are satisfied that the hazards involved are believed to be predictable. Physicians should cease any investigation if the hazards are found to outweigh the potential benefits.
8. In publication of the results of his or her research, the physician is obliged to preserve the accuracy of the results. Reports of experimentation not in accordance with the principles laid down in this Declaration should not be accepted for publication. 9. In any research on human beings, each potential subject must be adequately informed of the aims, methods, anticipated benefits and potential hazards of the study and the discomfort it may entail. He or she should be informed that he or she is at liberty to abstain from participation in the study and that he or she is free to withdraw his or her consent to participation at any time.
The physician should then obtain the subject's freely-given informed consent, preferably in writing.
10. When obtaining informed consent for the research project, the physician should be particularly cautious if the subject is in a dependent relationship to him or her or may consent under duress. In that case the informed consent should be obtained by a physician who is not engaged in the investigation and who is completely independent of this official relationship.
11. In case of legal incompetence, informed consent should be obtained from the legal guardian in accordance with national legislation. Where physical or mental incapacity makes it impossible to obtain informed consent, or when the subject is a minor, permission from the responsible relative replaces that of the subject in accordance with national legislation. Whenever the minor child is in fact able to give a consent, the minor's consent must be obtained in addition to the consent of the minor's legal guardian. The 0.78 mL vial of diluent contains sterile, preservative-free, pyrogen-free distilled water and is used for reconstitution of product supplied in 1 mL vials. The 6 mL vial of diluent contains sterile, pyrogen-free distilled water to which thimerosal (mercury derivative) 1:10,000 is added as a preservative. The 6 mL vial is for reconstitution of product supplied in 10 mL vials. After reconstitution with diluent as indicated on the label, the 0.5 mL dose is formulated to contain 50 µg of "isolated product" from each of Groups A, C, Y and W-135 in an isotonic sodium chloride solution.
Each dose of vaccine is also formulated to contain 2.5 mg to 5 mg of lactose added as a stabilizer 3. The vaccine when reconstituted is a clear colorless liquid.
Potency is evaluated by measuring the molecular size of each polysaccharide component using a column chromatography method as standardized by the US Food and Drug Administration (FDA) and the World Health Organization (WHO) 4 for Meningococcal Polysaccharide Vaccine.

CLINICAL PHARMACOLOGY
N. meningitidis causes both endemic and epidemic disease, principally meningitis and meningococcemia. As a result of the control of Haemophilus influenzae type b infections, N. meningitidis has become the leading cause of bacterial meningitis in children and young adults in the United States (US), with an estimated 2,600 cases each year. 5,6 The case-fatality rate is 13% for meningitis disease (defined as the isolation of N. meningitidis from cerebrospinal fluid) and 11.5% for persons who have N. meningitidis isolated from blood, 5,6 despite therapy with antimicrobial agents (e.g., penicillin) to which US strains remain clinically sensitive. 5 The incidence of meningococcal disease peaks in late winter to early spring. Based on multistate surveillance conducted during 1989 to 1991, serogroup B organisms accounted for 46% of all cases and serogroup C for 45%; serogroups W-135 and Y and strains that could not be serotyped accounted for most of the remaining cases. 5,6 Recent data indicate that the proportion of cases caused by serogroup Y strains is increasing. 5 In 1995, among the 30 states reporting supplemental data on culture-confirmed cases of meningococcal disease, serogroup Y accounted for 21% of cases. 7 Because of the success of H. influenzae type b vaccinations, the median age of persons with bacterial meningitis increased from 15 months in 1986 to 25 years in 1995. 8 The predominate organism causing meningitis in children 2 to 18 years of age is N. meningitidis based on 1995 surveillance data. 8 Serogroup A, which rarely causes disease in the US, is the most common cause of epidemics in Africa and Asia. A statewide serogroup B epidemic has been reported in the US. 9 Within the US, a vaccine for serogroup B is not yet available.
Outbreaks of serogroup C meningococcal disease (SCMD) have been occurring more frequently in the US since the early 1990s, and the use of vaccine to control these outbreaks has increased. 5 During 1980-1993, 21 outbreaks of SCMD were identified; eight of these occurred during 1992-1993. 10 Each of these 21 outbreaks involved from three to 45 cases of SCMD, and most outbreaks had attack rates exceeding 10 cases per 100,000 population, which is approximately 20 times higher than rates of endemic SCMD. 5 During 1981-1988, only 7,600 doses of Confidential 40 Confidential meningococcal vaccine were used to control four outbreaks; whereas, from January 1992 through June 1993, 180,000 doses of vaccine were used in response to eight outbreaks. 5 Several discoveries impacted the future of meningococcal polysaccharide vaccines and demonstrated the significance of anti-capsular antibodies in protection. 11 In the late 1930s, serogroup-specific antigens of meningococcal serogroups A and C were identified as polysaccharides. 9 During the mid 1940s, investigators demonstrated that the protection of mice by anti-serogroup A meningococcal horse serum was directly related to its content of antipolysaccharide antibodies. 11 Meningococcal polysaccharide vaccines were first demonstrated to be immunogenic in humans by Gotschlich and his co-workers in the 1960s when immunization of US Army recruits with serogroup A and C polysaccharides induced protective antibodies. 11 The investigators recorded a significantly reduced acquisition rate of serogroup C carriage among vaccinated recruits compared with unvaccinated individuals. 11 Persons who have certain medical conditions are at increased risk for developing meningococcal infection. Meningococcal disease is particularly common among persons who have component deficiencies in the terminal common complement pathway (C3, C5-C9); many of these persons experience multiple episodes of infection. 5 Asplenic persons also may be at increased risk for acquiring meningococcal disease with particularly severe infections. 5 Persons who have other diseases associated with immunosuppression (e.g., human immunodeficiency virus [HIV] and Streptococcus pneumoniae) may be at higher risk for developing meningococcal disease and for disease caused by some other encapsulated bacteria. 5 Evidence suggests that HIV-infected persons are not at substantially increased risk for epidemic serogroup A meningococcal disease; 5 however, such patients may be at increased risk for sporadic meningococcal disease or disease caused by other meningococcal serogroups. 5 Previously, military recruits had high rates of meningococcal disease, particularly serogroup C disease; however, since the initiation of routine vaccination of recruits with bivalent A/C meningococcal vaccine in 1971, the high rates of meningococcal disease caused by those serogroups have decreased substantially and cases occur infrequently. 5 A retrospective, epidemiological study was conducted in Maryland to compare the incidence of invasive meningococcal infection in college students with that of the general population of the same age. For the years 1992 to 1997, the incidence of meningococcal infection in Maryland college students was similar to the incidence of the general Maryland population of the same age. However, college students residing on-campus appeared to be at higher risk than those residing off campus. 12 Vaccine efficacy. The immunogenicity and clinical efficacy of serogroups A and C meningococcal vaccines have been well established. 5 The serogroup A polysaccharide induces antibody in some children as young as 3 months of age, although a response comparable with that among adults is not achieved until 4 or 5 years of age; the serogroup C component is poorly immunogenic in recipients who are less than 18 to 24 months of age. 5 The serogroups A and C vaccines have demonstrated estimated clinical efficacies of 85% to 100% in older children and adults and are useful in controlling epidemics. 5 Serogroups Y and W-135 polysaccharides are safe and immunogenic in adults and in children greater than 2 years of age. 5 Although clinical protection has not been documented, vaccination with these polysaccharides induces bactericidal antibody. The antibody responses to each of the four polysaccharides in the quadrivalent vaccine are serogroup-specific and independent. 5 Efficacy of serogroup A meningococcal vaccines was demonstrated in the 1970s in Africa and Finland, Egyptian school children aged 6 to 15 years showed 90% or greater protection during the first year after immunization with two different molecular sizes of serogroup A polysaccharide. 11 The higher molecular weight vaccine provided protection for at least three years. 11 In Finland, a randomized controlled mass immunization trial with serogroup A vaccine was conducted in response to a serogroup A epidemic. Results indicated 90 to 100% protection for three years. 11 In Rwanda, vaccination with bivalent A/C polysaccharide vaccine was performed in response to a serogroup A epidemic. A complete cessation of meningococcal disease was observed within two weeks of vaccination, yet the serogroup A carrier rate remained unchanged. 11 Efficacy of serogroup C meningococcal vaccines was demonstrated in a field trial involving 20,000 troops in the US Army. Results suggested 90% efficacy under epidemic conditions which existed in basic training centers. 13 In Brazil, young children were vaccinated with serogroup C polysaccharide in response to a serogroup C epidemic. Results indicated that the vaccine was not effective in children under 24 months of age and only 52% effective in children aged 24 to 36 months. 11 However, studies suggested that the vaccine used in this trial was less immunogenic than other batches of similar vaccine that were used in US children; also, it was shown that the molecular size of the vaccine was smaller than the serogroup C polysaccharide in the present vaccine. 13 Thus, it is quite probable that the current serogroup C polysaccharide vaccine is more effective. 11 A study performed using 4 lots of Menomune ® -A/C/Y/W-135 in 150 adults showed at least a 4-fold increase in bactericidal antibodies to all groups in greater than 90 percent of the subjects. 14,15 A study was conducted in 73 children 2 to 12 years of age. Post-immunization sera were not obtained on four children; seroconversion rates were calculated on 69 paired samples. Seroconversion rates as measured by bactericidal antibody were: Group A -72%, Group C -58%, Group Y -90% and Group W-135 -82%. Seroconversion rates as measured by a 2-fold rise in antibody titers based on Solid Phase Radioimmunoassay were: Group A -99%, Group C -99%, Group Y -97% and Group W-135 -89%. 16 Duration of efficacy. Measurable levels of antibodies against the group A and C polysaccharides decrease markedly during the first 3 years following a single dose of vaccine. 5 This decrease in antibody occurs more rapidly in infants and young children than in adults. Similarly, although vaccine-induced clinical protection probably persists in schoolchildren and adults for at least 3 years, the efficacy of the group A vaccine in young children may decrease markedly with the passage of time. In a 3-year study, efficacy declined from greater than 90% to less than 10% among children who were less than 4 years of age at the time of vaccination, whereas among children who were greater than or equal to 4 years of age when vaccinated, efficacy was 67% 3 years later. 5 The American College Health Association (ACHA) also recommends that college students consider vaccination to reduce the risk for potentially fatal meningococcal disease. 19 Vaccinations also should be considered for household or institutional contacts of persons with meningococcal disease and for medical and laboratory personnel at risk of exposure to meningococcal disease.
This vaccine will not stimulate protection against infections caused by organisms other than Groups A, C, Y and W-135 meningococci.
Protective antibody levels may be achieved within 7 to 10 days after vaccination. 5 Menomune ® -A/C/Y/W-135 vaccine is not to be used for treatment of actual infection.
Menomune ® -A/C/Y/W-135 vaccine will not protect against other etiologic agents, including N. meningitidis serogroup B, that cause meningitis.
Menomune ® -A/C/Y/W-135 vaccine is not indicated for infants and children younger than 2 years of age except as short-term protection of infants 3 months and older against Group A. 11 As with any vaccine, vaccination with Menomune ® -A/C/Y/W-135 may not protect 100% of susceptible individuals.
For persons remaining at high-risk, especially children who were first vaccinated at < 4 years of age, revaccination may be indicated. 5  Confidential CONTRAINDICATIONS Immunization should be deferred during the course of any acute illness. A CONTRAINDICATION TO ADMINISTER MENOMUNE ® -A/C/Y/W-135 TO INDIVIDUALS  KNOWN TO BE SENSITIVE TO THIMEROSAL OR ANY OTHER COMPONENT OF THE VACCINE. FOR  INDIVIDUALS SENSITIVE TO THIMEROSAL, ADMINISTER THE ONE DOSE PACKAGE SIZE AND  RECONSTITUTE WITH THE 0.78 ML VIAL OF DILUENT THAT CONTAINS NO PRESERVATIVE. WARNING This product contains dry natural latex rubber as follows: The stopper to the vial contains dry natural latex rubber.

IT IS
If the vaccine is used in persons receiving immunosuppressive therapy, the expected immune response may not be obtained.
Menomune ® -A/C/Y/W-135 should NOT be given at the same time as whole-cell pertussis or whole-cell typhoid vaccines due to combined endotoxin content. 0,221.

PRECAUTIONS
GENERAL Care is to be taken by the health-care provider for the safe and effective use of Menomune ® -A/C/Y/W-135.

EPINEPHRINE INJECTION (1:1000) MUST BE IMMEDIATELY AVAILABLE TO COMBAT UNEXPECTED ANAPHYLACTIC OR OTHER ALLERGIC REACTIONS.
Prior to an injection of any vaccine, all known precautions should be taken to prevent adverse reactions. This includes a review of the patient's history with respect to possible sensitivity to the vaccine or similar vaccines and to possible sensitivity to dry natural latex rubber.
Special care should be taken to avoid injecting the vaccine intradermally, intramuscularly, or intravenously since clinical studies have not been done to establish safety and efficacy of the vaccine using these routes of administration.
Health-care providers should obtain the previous immunization history of the vaccinee, and inquire about the current health status of the vaccinee.
A separate, sterile syringe and needle or a sterile disposable unit should be used for each patient to prevent transmission of hepatitis and other infectious agents from person to person. Needles should not be recapped and should be disposed of according to biohazard waste guidelines.
INFORMATION FOR PATIENT Patients, parents or guardians should be fully informed of the benefits and risks of immunization with Menomune ® -A/C/Y/W-135.
Patients, parents or guardians should be instructed to report any serious adverse reactions to their health-care provider.
As part of the patient's immunization record, the date, lot number and manufacturer of the vaccine administered should be recorded. 22

ADVERSE REACTIONS
Adverse reactions to meningococcal vaccine are mild and consist principally of pain and redness at the injection site for 1 to 2 days. Pain at the site of injection is the most commonly reported adverse reaction, and a transient fever might develop in less than or equal to 2% of young children. 5 Adverse events reported by 150 adults following vaccination with Menomune ® -A/C/Y/W-135 are shown in Table  1. 14  In a clinical study involving 73 children 2 to 12 years of age, who received Menomune ® -A/C/Y/W-135, local reactions consisting of erythema or tenderness were seen in approximately 40% of the children. 15 In another clinical study involving 53 children 4 to 6 years of age, who received Menomune ® -A/C/Y/W-135, erythema was seen in 89% of the children, swelling in 92% and tenderness in 64%. None of these reactions were considered serious or necessitated medical intervention. 26 On rare occasions, IgA nephropathy has occurred following vaccinations with Menomune ® -A/C/Y/W-135. However, a cause and effect relationship has not been established. 16 Menomune ® -A/C/Y/W-135 should NOT be given at the same time as whole-cell pertussis or whole-cell typhoid vaccines due to combined endotoxin content. 20,21 As with the administration of any vaccine, vaccine components can cause hypersensitivity reactions in some recipients.

Reporting of Adverse Events
The National Vaccine Injury Compensation Program, established by the National Childhood Vaccine Injury Act of 1986, requires physicians and other health-care providers who administer vaccines to maintain permanent vaccination records and to report occurrences of certain adverse events to the US Department of Health and Human Services. Reportable events include those listed in the Act for each vaccine and events specified in the package insert as contraindications to further doses of that vaccine. 22,23,24 Reporting by patients, parents or guardians of all adverse events occurring after vaccine administration should be encouraged. Adverse events following immunization with vaccine should be reported by the health-care provider to the US Department of Health and Human Services (DHHS) Vaccine Adverse Event Reporting Systems (VAERS).
Reporting forms and information about reporting requirements or completion of the form can be obtained from VAERS through a toll-free number 1-800-822-7967. 24

Confidential
Health-care providers also should report these events to the Pharmacovigilance Department, Aventis Pasteur Inc., Discovery Drive, Swiftwater, PA 18370 or call 1-800-822-2463.

DOSAGE AND ADMINISTRATION
Parenteral drug products should be inspected visually for extraneous particulate matter and/or discoloration prior to administration whenever solution and container permit. If either of these conditions exist, the vaccine should not be administered.
Reconstitute the vaccine using only the diluent supplied for this purpose. Draw the volume of diluent shown on the diluent label into a suitable size syringe and inject into the vial containing the vaccine. Shake vial until the vaccine is dissolved.
The immunizing dose is a single injection of 0.5 mL administered subcutaneously.
Special care should be taken to avoid injecting the vaccine intradermally, intramuscularly, or intravenously since clinical studies have not been done to establish safety and efficacy of the vaccine using these routes of administration.

Primary Immunization
For both adults and children, vaccine is administered subcutaneously as a single 0.5 mL dose. Protective antibody levels may be achieved within 7 to 10 days after vaccination. 5

REVACCINATION
Revaccination of a single 0.5 mL dose administered subcutaneously may be indicated for individuals at high-risk of infection, particularly children who were first vaccinated when they were less than 4 years of age; such children should be considered for revaccination after 2 or 3 years if they remain at high-risk. Although the need for revaccination in older children and adults has not been determined, antibody levels decline rapidly over 2 to 3 years, and if indications still exist for immunization, revaccination may be considered within 3 to 5 years. 5,18 Simultaneous administration of Menomune ® -A/C/Y/W-135 can be given concurrently with other vaccines at separate sites and separate syringes. 27 However, due to the combined endotoxin content, the vaccine should NOT be administered at the same time as whole-cell pertussis or whole-cell typhoid vaccines. 20    Not to be quoted or distributed Confidential