Meningococcal ACWYX Conjugate Vaccine in 2-to-29-Year-Olds in Mali and Gambia.

BACKGROUND: An effective, affordable, multivalent meningococcal conjugate vaccine is needed to prevent epidemic meningitis in the African meningitis belt. Data on the safety and immunogenicity of NmCV-5, a pentavalent vaccine targeting the A, C, W, Y, and X serogroups, have been limited. METHODS: We conducted a phase 3, noninferiority trial involving healthy 2-to-29-year-olds in Mali and Gambia. Participants were randomly assigned in a 2:1 ratio to receive a single intramuscular dose of NmCV-5 or the quadrivalent vaccine MenACWY-D. Immunogenicity was assessed at day 28. The noninferiority of NmCV-5 to MenACWY-D was assessed on the basis of the difference in the percentage of participants with a seroresponse (defined as prespecified changes in titer; margin, lower limit of the 96% confidence interval [CI] above -10 percentage points) or geometric mean titer (GMT) ratios (margin, lower limit of the 98.98% CI >0.5). Serogroup X responses in the NmCV-5 group were compared with the lowest response among the MenACWY-D serogroups. Safety was also assessed. RESULTS: A total of 1800 participants received NmCV-5 or MenACWY-D. In the NmCV-5 group, the percentage of participants with a seroresponse ranged from 70.5% (95% CI, 67.8 to 73.2) for serogroup A to 98.5% (95% CI, 97.6 to 99.2) for serogroup W; the percentage with a serogroup X response was 97.2% (95% CI, 96.0 to 98.1). The overall difference between the two vaccines in seroresponse for the four shared serogroups ranged from 1.2 percentage points (96% CI, -0.3 to 3.1) for serogroup W to 20.5 percentage points (96% CI, 15.4 to 25.6) for serogroup A. The overall GMT ratios for the four shared serogroups ranged from 1.7 (98.98% CI, 1.5 to 1.9) for serogroup A to 2.8 (98.98% CI, 2.3 to 3.5) for serogroup C. The serogroup X component of the NmCV-5 vaccine generated seroresponses and GMTs that met the prespecified noninferiority criteria. The incidence of systemic adverse events was similar in the two groups (11.1% in the NmCV-5 group and 9.2% in the MenACWY-D group). CONCLUSIONS: For all four serotypes in common with the MenACWY-D vaccine, the NmCV-5 vaccine elicited immune responses that were noninferior to those elicited by the MenACWY-D vaccine. NmCV-5 also elicited immune responses to serogroup X. No safety concerns were evident. (Funded by the U.K. Foreign, Commonwealth, and Development Office and others; ClinicalTrials.gov number, NCT03964012.).

Immunogenicity and safety of a 10-valent pneumococcal conjugate vaccine administered as a 2 + 1 schedule to healthy infants in The Gambia: a single-centre, double-blind, active-controlled, randomised, phase 3 trial.

BACKGROUND: Three pneumococcal conjugate vaccines (PCVs) are currently licensed and WHO prequalified for supply by UN agencies. Here, we aimed to investigate the safety and immunogenicity of SIIPL-PCV compared with PHiD-CV and PCV13, when administered to infants according to a 2 + 1 schedule. METHODS: This single-centre, double-blind, active-controlled, randomised, phase 3 trial was done in Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine clinical trial facilities within two government health centres in the western region of The Gambia. Healthy, PCV-naive infants aged 6-8 weeks were enrolled if they weighed at least 3·5 kg and had no clinically significant health complaints, as determined by history and clinical examination. Eligible infants were randomly assigned (1:1:1) to receive either SIIPL-PCV, PHiD-CV, or PCV13 using permuted blocks of variable size. Parents and the trial staff assessing all study outcomes were masked to vaccine group. The first PCV vaccine was given with other routine Expanded Programme on Immunization vaccines when infants were aged 6-8 weeks (visit 1). At visit 2, routine vaccines alone (without a PCV) were administered. At visit 3, the second dose of the PCV was administered alongside other routine vaccines. At visit 4, a blood sample was collected. Visits 1-4 took place at intervals of 4 weeks. The booster PCV was administered at age 9-18 months (visit 5), with final follow-up 4 weeks after the booster (visit 6). The primary immunogenicity outcome compared the serotype-specific IgG geometric mean concentrations (GMCs) generated by SIIPL-PCV with those generated by PHiD-CV and PCV13, 4 weeks after the booster. We used descriptive 95% CIs without adjustment for multiplicity. Immunogenicity analyses were done in the per protocol population (defined as all children who received all the assigned study vaccines, who had an immunogenicity measurement available, and who had no protocol deviations that might interfere with the immunogenicity assessment). This trial was registered with the Pan African Clinical Trials Registry, PACTR201907754270299, and ClinicalTrials.gov, NCT03896477. FINDINGS: Between July 18 and Nov 14, 2019, 745 infants were assessed for study eligibility. Of these, 85 infants (11%) were ineligible and 660 (89%) were enrolled and randomly assigned to receive SIIPL-PCV (n=220), PHiD-CV (n=220), or PCV13 (n=220). 602 infants (91%) were included in the per protocol immunogenicity population. The median age at vaccination was 46 days (range 42-56). 342 infants (52%) were female and 318 (48%) were male. Post-booster serotype-specific IgG GMCs generated by SIIPL-PCV ranged from 1·54 µg/mL (95% CI 1·38-1·73) for serotype 5 to 12·46 µg/mL (11·07-14·01) for serotype 6B. Post-booster GMCs against shared serotypes generated by PHiD-CV ranged from 0·80 µg/mL (0·72-0·88) for serotype 5 to 17·31 µg/mL (14·83-20·20) for serotype 19F. Post-booster GMCs generated by PCV13 ranged from 2·04 µg/mL (1·86-2·24) for serotype 5 to 15·54 µg/mL (13·71-17·60) for serotype 6B. Post-booster IgG GMCs generated by SIIPL-PCV were higher than those generated by PHiD-CV for seven of the eight shared serotypes (1, 5, 6B, 7F, 9V, 14, and 23F). The GMC generated by serotype 19F was higher after PHiD-CV. The SIIPL-PCV to PHiD-CV GMC ratios for shared serotypes ranged from 0·64 (95% CI 0·52-0·79) for serotype 19F to 2·91 (2·47-3·44) for serotype 1. The serotype 1 GMC generated by SIIPL-PCV was higher than that generated by PCV13, whereas serotype 5, 6A, 19A, and 19F GMCs were higher after PCV13. The SIIPL-PCV to PCV13 GMC ratios ranged from 0·72 (0·60-0·87) for serotype 19A to 1·44 (1·23-1·69) for serotype 1. INTERPRETATION: SIIPL-PCV was safe and immunogenic when given to infants in The Gambia according to a 2 + 1 schedule. This PCV is expected to provide similar protection against invasive and mucosal pneumococcal disease to the protection provided by PCV13 and PHiD-CV, for which effectiveness data are available. Generating post-implementation data on the impact of SIIPL-PCV on pneumococcal disease endpoints remains important. FUNDING: Bill & Melinda Gates Foundation.

RSV neutralization assays - Use in immune response assessment.

Respiratory syncytial virus (RSV) is a leading cause of respiratory illness among children and infants worldwide, yet no licensed vaccine exists to reduce the risk of disease. At least 16 RSV vaccine candidates are currently in clinical development and many are designed to induce robust virus neutralizing immune responses. RSV neutralizing antibody (nAb)-mediated interventions such as intravenous immunoglobulin (IVIG) and palivizumab provide passive protection against serious lower respiratory tract disease due to RSV, validating nAbs as a correlate of protection. To identify correlates of protection for vaccine candidates that have demonstrated their protective efficacy, an investigator can use assays designed to measure nAb responses. However, there is no standard method of measurement; individual laboratories have developed their own methods to measure the ability of nAbs to reduce the infectivity of a defined virus dose in a variety of cell lines, leading to establishment of the broad variety of RSV neutralization assay formats currently in use. Standardizing the RSV neutralization assay is an essential step toward better assessment of nAb responses to vaccine candidates. Use of a common reference standard by all makes comparing the immunogenicity of different vaccine candidates feasible. In the context of vaccine development, the WHO 1st International Standard for Antiserum to RSV (RSV IS) has been shown to be suitable for harmonizing results across laboratories and assay formats used to measure nAb titers to RSV/A and RSV/B in human sera. This review describes the broad variety of RSV virus neutralization assay formats currently in use and the importance of the RSV IS for harmonization of results across formats and across laboratories. It also outlines good practices for key assay components and data analysis to promote the quality and consistency of measuring RSV nAb titers in serum specimens.

Meningococcal Serogroup ACWYX Conjugate Vaccine in Malian Toddlers.

BACKGROUND: Neisseria meningitidis serogroups A, B, C, W, X, and Y cause outbreaks of meningococcal disease. Quadrivalent conjugate vaccines targeting the A, C, W, and Y serogroups are available. A pentavalent vaccine that also includes serogroup X (NmCV-5) is under development. METHODS: We conducted a phase 2, observer-blinded, randomized, controlled trial involving Malian children 12 to 16 months of age. Participants were assigned in a 2:2:1 ratio to receive nonadjuvanted NmCV-5, alum-adjuvanted NmCV-5, or the quadrivalent vaccine MenACWY-D, administered intramuscularly in two doses 12 weeks apart. Participants were followed for safety for 169 days. Immunogenicity was assessed with an assay for serum bactericidal antibody (SBA) with rabbit complement on days 0, 28, 84, and 112. RESULTS: A total of 376 participants underwent randomization, with 150 assigned to each NmCV-5 group and 76 to the MenACWY-D group; 362 participants received both doses of vaccine. A total of 1% of the participants in the nonadjuvanted NmCV-5 group, 1% of those in the adjuvanted NmCV-5 group, and 4% of those in the MenACWY-D group reported local solicited adverse events; 6%, 5%, and 7% of the participants, respectively, reported systemic solicited adverse events. An SBA titer of at least 128 was seen in 91 to 100% (for all five serotypes) of the participants in the NmCV-5 groups and in 36 to 99% (excluding serogroup X) of those in the MenACWY-D group at day 84 (before the second dose); the same threshold was met in 99 to 100% (for all five serotypes) of the participants in the NmCV-5 groups and in 92 to 100% (excluding serogroup X) of those in the MenACWY-D group at day 112. Immune responses to the nonadjuvanted and adjuvanted NmCV-5 formulations were similar. CONCLUSIONS: No safety concerns were identified with two doses of NmCV-5. A single dose of NmCV-5 elicited immune responses that were similar to those observed with two doses of MenACWY-D. Adjuvanted NmCV-5 provided no discernible benefit over nonadjuvanted NmCV-5. (Funded by the U.K. Foreign, Commonwealth, and Development Office; ClinicalTrials.gov number, NCT03295318.).

Immunogenicity and safety of a novel ten-valent pneumococcal conjugate vaccine in healthy infants in The Gambia: a phase 3, randomised, double-blind, non-inferiority trial.

BACKGROUND: An affordable pneumococcal conjugate vaccine (PCV) is needed to ensure sustainable access in low-income and middle-income countries. This trial examined the immunogenicity and safety of a novel ten-valent PCV (SIIPL-PCV) containing serotypes 1, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F, and 23F compared with the pneumococcal polysaccharide protein D-conjugate vaccine (PHiD-CV; Synflorix; GlaxoSmithKline; Brentford, UK). METHODS: In this single-centre, randomised, double-blind, phase 3, non-inferiority trial in The Gambia, healthy, PCV-naive infants aged 6-8 weeks were enrolled and assigned using permuted block randomisation to receive one of three lots of SIIPL-PCV or to PHiD-CV in a ratio of 2:2:2:3. Parents and all staff assessing study outcomes were masked to group assignment. Vaccines (0·5 mL SIIPL-PCV or 0·5 mL PHiD-CV) were administered at ages 6, 10, and 14 weeks by intramuscular injection. Primary immunogenicity outcomes, measured at age 18 weeks, were serotype-specific IgG geometric mean concentrations (GMCs) and seroresponse rates (IgG ≥ 0·35 µg/mL). Lot-to-lot equivalence (objective 1) was shown if the upper and lower bounds of the two-sided 95% CI around the GMC ratio for each pairwise lot-to-lot comparison was between the 0·5 and 2·0 equivalence margins for all ten serotypes. The immunogenicity of SIIPL-PCV was defined as being non-inferior to that of PHiD-CV (objective 2) if, for at least seven of the ten serotypes in SIIPL-PCV, the lower bound of the 97·5% CI for the GMC ratio was greater than 0·5, or the lower bound of the 97·5% CI for differences in seroresponse rate was greater than -10%. The GMC and seroresponse rates to serotypes 6A and 19A, which are not in PHiD-CV, were compared with those of the serotype in PHiD-CV that had the lowest seroresponse rate. Non-inferiority of the immune responses to antigens in the co-administered Expanded Programme on Immunization (EPI) vaccines (objective 3) was declared if the lower bound of the 95% CI for the difference between SIIPL-PCV and PHiD-CV in seroresponse rates, or GMC ratios for pertussis antigens, was greater than -10% (or 0·5 for pertussis antigens) for all vaccine antigens. Safety data were assessed according to treatment received at the first visit in infants who received at least one dose of study vaccine and for whom at least some post-vaccination safety data were available. The primary immunogenicity analysis was in the per-protocol immunogenicity population, which included infants who received all study vaccines and had immunogenicity measurements after vaccination and no major protocol deviations. This trial is registered at ClinicalTrials.gov (NCT03197376). FINDINGS: Between June 21, 2017, and Jan 29, 2018, 2250 infants were enrolled and randomly assigned to receive SIIPL-PCV (n=1503; 502 to lot 1, 501 to lot 2, and 500 to lot 3) or PHiD-CV (n=747). 1458 (97·0%) infants assigned to SIIPL-PCV and 724 (96·9%) assigned to PHiD-CV were included in the per-protocol primary immunogenicity analysis. Lot-to-lot equivalence was shown, with the lowest lower bound of the 95% CI for the GMC ratio being 0·52 (for serotype 6B in lot 2 vs lot 3) and the highest upper bound being 1·69 (for serotype 6B in lot 1 vs lot 2). SIIPL-PCV was non-inferior to PHiD-CV in terms of immunogenicity: the lower bound of the 97·5% CI for the GMC ratio was greater than 0·5 (the lowest being 0·67 for serotype 19F) and the lower bound of the 97·5% CI for the difference in seroresponse rate was greater than -10% (the lowest being -2·2% for serotype 6B) for all ten serotypes in SIIPL-PCV. The lowest seroresponse rate after PHiD-CV was to serotype 6B (76·7% [95% CI 73·4-79·7]). This serotype was therefore used for the comparisons with serotype 6A and 19A in SIIPL-PCV. Non-inferiority of immune responses to the EPI vaccines after co-administration with SIIPL-PCV compared with after co-administration with PHiD-CV was shown for all vaccine antigens included in the primary series. The lowest lower bound of the 95% CI for the difference in seroresponse rates was -7·1% for rotavirus antibody and for the GMC ratio for pertussis antigens was 0·62 for anti-pertussis toxoid. 1131 (75·2%) of 1503 infants in the SIIPL-PCV group and 572 (76·6%) of 747 in the PHiD-CV group had at least one unsolicited adverse event. 36 (2·4%) participants in the SIIPL-PCV group and 18 (2·4%) in the PHiD-CV group had a serious adverse event; none were considered related to vaccination. In infants who were selected to have solicited adverse events recorded, injection-site induration after primary vaccinations occurred in 27 (4·9%) of 751 infants who received SIIPL-PCV versus 34 (9·4%) of 364 who received PHiD-CV (p=0·0032). There were no other notable differences in the safety profiles of the two vaccines. One infant in the SIIPL-PCV group and two in the PHiD-CV group died during the study. The deaths were not considered to be related to study vaccination or study participation. INTERPRETATION: The immunogenicity of SIIPL-PCV was non-inferior to that of PHiD-CV, for which efficacy and effectiveness data against pneumococcal disease are available. The vaccine is safe and can be co-administered with routine EPI vaccines. The data generated in this trial have supported the licensure and pre-qualification of SIIPL-PCV, making the vaccine available for introduction into national immunisation programmes. Generating post-implementation data confirming vaccine impact remains important. FUNDING: Bill & Melinda Gates Foundation.

A Randomized, Double-Blinded, Placebo-Controlled, Phase 1 Study of a Replication-Defective Herpes Simplex Virus (HSV) Type 2 Vaccine, HSV529, in Adults With or Without HSV Infection.

BACKGROUND: Herpes simplex virus 2 (HSV2) causes genital herpes in >400 million persons worldwide. METHODS: We conducted a randomized, double-blinded, placebo-controlled trial of a replication-defective HSV2 vaccine, HSV529. Twenty adults were enrolled in each of 3 serogroups of individuals: those negative for both HSV1 and HSV2 (HSV1-/HSV2-), those positive or negative for HSV1 and positive for HSV2 (HSV1±/HSV2+), and those positive for HSV1 and negative for HSV2 (HSV1+/HSV2-). Sixty participants received vaccine or placebo at 0, 1, and 6 months. The primary end point was the frequency of solicited local and systemic reactions to vaccination. RESULTS: Eighty-nine percent of vaccinees experienced mild-to-moderate solicited injection site reactions, compared with 47% of placebo recipients (95% confidence interval [CI], 12.9%-67.6%; P = .006). Sixty-four percent of vaccinees experienced systemic reactions, compared with 53% of placebo recipients (95% CI, -17.9% to 40.2%; P = .44). Seventy-eight percent of HSV1-/HSV2- vaccine recipients had a ≥4-fold increase in neutralizing antibody titer after 3 doses of vaccine, whereas none of the participants in the other serogroups had such responses. HSV2-specific CD4+ T-cell responses were detected in 36%, 46%, and 27% of HSV1-/HSV2-, HSV1±/HSV2+, and HSV1+/HSV2- participants, respectively, 1 month after the third dose of vaccine, and CD8+ T-cell responses were detected in 14%, 8%, and 18% of participants, respectively. CONCLUSIONS: HSV529 vaccine was safe and elicited neutralizing antibody and modest CD4+ T-cell responses in HSV-seronegative vaccinees. CLINICAL TRIALS REGISTRATION: NCT01915212.

Safety and immunogenicity of a pentavalent meningococcal conjugate vaccine containing serogroups A, C, Y, W, and X in healthy adults: a phase 1, single-centre, double-blind, randomised, controlled study.

BACKGROUND: Invasive meningococcal disease is an important public health problem, especially in sub-Saharan Africa. After introduction of MenAfriVac in 2010, Neisseria meningitidis serogroup A disease has been almost eliminated from the region. However, serogroups C, W, Y, and X continue to cause disease outbreaks. We assessed the NmCV-5 pentavalent meningococcal conjugate vaccine targeting A, C, Y, W, and X serogroups in a first-in-man, phase 1 study. METHODS: We did a single-centre, double-blind, randomised controlled trial at a research clinic in Baltimore (MD, USA). Participants were healthy adults aged 18-45 years with no history of meningococcal vaccination or previous meningococcal infection. We randomly assigned participants (1:1:1) by an SAS-generated random schedule to a single, 0·5 mL, intramuscular injection of aluminium-phosphate adjuvanted NmCV-5, non-adjuvanted NmCV-5, or control (the quadrivalent meningococcal conjugate vaccine Menactra). The randomisation sequence used a permuted block design with randomly chosen block sizes of three and six. The vaccines were prepared, labelled, and administered with procedures to ensure participants and study personnel remained masked to treatment. After vaccination, participants were observed in the clinic for 60 min for adverse reactions. Participants recorded daily temperature and injection site or systemic reactions at home and returned to the clinic for follow-up visits on days 7, 28, and 84 for safety assessments; blood samples were also collected on day 7 for safety laboratory assessment. A phone call contact was made 6 months after vaccination. Serum was collected before vaccination and 28 days after vaccination for immunological assessment with a rabbit complement-dependent serum bactericidal antibody (rSBA) assay. The primary objective was an intention-to-treat assessment of safety, measuring local and systemic reactogenicity over 7 days, unsolicited adverse events through 28 days, and serious adverse events over 6 months. The secondary objective for the assessment of immunogenicity, was a per-protocol analysis of rSBA before and 28 days after vaccination. This trial is registered with ClinicalTrials.gov, number NCT02810340. FINDINGS: Between Aug 17, 2016, and Feb 16, 2017, we assigned 20 participants to each vaccine. All vaccines were well-tolerated. Pain was the most common local reaction, occurring in 12 (60%), ten (50%), and seven (35%) participants in the adjuvanted NmCV-5, non-adjuvanted NmCV-5, and control groups, respectively. Headache was the most common systemic reaction, occurring in five (25%), three (15%), and three (15%), respectively. Most solicited reactogenicity adverse reactions were mild (60 [74%] of 81) and all were self-limiting. None of the differences in proportions of individuals with each solicited reaction was significant (p>0·300 for all comparisons) between the three vaccination groups. There were no serious adverse events and 19 unsolicited non-serious adverse events in 14 (23%) participants. Both adjuvanted and non-adjuvanted NmCV-5 elicited high rSBA titres against all five meningococcal serogroups. The pre-vaccination geometric mean titres (GMTs) ranged from 3·36 to 53·80 for the control, from 6·28 to 187·00 for the adjuvanted vaccine, and from 4·29 to 350·00 for the non-adjuvanted vaccine, and the post-vaccination GMT ranged from 3·14 to 3214 for the control, from 1351 to 8192 for the adjuvanted vaccine, and from 1607 to 11 191 for the non-adjuvanted vaccine. Predicted seroprotective responses (ie, an increase in rSBA titres of eight times or more) for the adjuvanted and non-adjuvanted NmCV-5 were similar to control responses for all five serogroups. INTERPRETATION: The adjuvanted and non-adjuvanted NmCV-5 vaccines were well tolerated and did not produce concerning adverse effects and resulted in immune responses that are predicted to confer protection against all five targeted serogroups of invasive meningococcal disease. Further clinical testing of NmCV-5 is ongoing, and additional clinical trials are necessary to confirm the safety and immunogenicity of NmCV-5 in target populations. FUNDING: UK Department for International Development.

A multi-laboratory study of diverse RSV neutralization assays indicates feasibility for harmonization with an international standard.

A current barrier to the standardized evaluation of respiratory syncytial virus (RSV) vaccine candidates is the wide variety of virus neutralization assay formats currently in use for assessing immunogenicity. Assay formats vary widely in labor intensiveness, duration, and sample throughput. Furthermore, the cell lines and virus strains used are not consistent among formats. The purpose of this multi-laboratory study was to assess the variability across a diverse array of assay formats that quantitate RSV neutralizing antibodies. Using a common specimen panel, the degree of overall agreement among existing assays was evaluated to inform on the need for harmonization of assay results. A total of 12 laboratories participated in the blinded survey study by testing a panel comprised of 57 samples chosen to span the reportable titer range of the assays. An independent statistical analysis was conducted to measure overall agreement of assay results. This analysis showed that precision was consistently high, whereas agreement varied widely among assays. To examine whether agreement could be improved, we conducted a harmonization exercise using a variety of sample types as pseudo standards. The results showed that the level of agreement could be improved, and provided information on the suitability of samples for developing an international standard.

Stability of live attenuated rotavirus vaccine with selected preservatives and primary containers.

Rotavirus infection, which can be prevented by vaccination, is responsible for a high burden of acute gastroenteritis disease in children, especially in low-income countries. An appropriate formulation, packaging, and delivery device for oral rotavirus vaccine has the potential to reduce the manufacturing cost of the vaccine and the logistical impact associated with introduction of a new vaccine, simplify the vaccination procedure, and ensure that the vaccine is safely and accurately delivered to children. Single-dose prefilled presentations can be easy to use; however, they are typically more expensive, can be a bottleneck during production, and occupy a greater volume per dose vis-à-vis supply chain storage and medical waste disposal, which is a challenge in low-resource settings. Multi-dose presentations used thus far have other issues, including increased wastage of vaccine and the need for separate delivery devices. In this study, the goals were to evaluate both the technical feasibility of using preservatives to develop a liquid multi-dose formulation and the primary packaging alternatives for orally delivered, liquid rotavirus vaccines. The feasibility evaluation included evaluation of commonly used preservatives for compatibility with rotavirus vaccines and stability testing of rotavirus vaccine in various primary containers, including Lameplast's plastic tubes, BD's oral dispenser version of Uniject™ (Uniject DP), rommelag's blow-fill-seal containers, and MEDInstill's multi-dose vial and pouch. These presentations were compared to a standard glass vial. The results showed that none of the preservatives tested were compatible with a live attenuated rotavirus vaccine because they had a detrimental effect on the viability of the virus. In the presence of preservatives, vaccine virus titers declined to undetectable levels within 1 month. The vaccine formulation without preservatives maintained a stability profile over 12 months in all primary containers that was similar to its profile in standard glass vials. This study demonstrates that there are multiple options for the primary container for rotavirus vaccines intended for oral delivery. Selection of an optimal primary container should take into consideration additional factors, including stability as well as cold chain volume, usability, cost, and manufacturing feasibility.

Serum and mucosal antibody responses to inactivated polio vaccine after sublingual immunization using a thermoresponsive gel delivery system.

Administering vaccines directly to mucosal surfaces can induce both serum and mucosal immune responses. Mucosal responses may prevent establishment of initial infection at the port of entry and subsequent dissemination to other sites. The sublingual route is attractive for mucosal vaccination, but both a safe, potent adjuvant and a novel formulation are needed to achieve an adequate immune response. We report the use of a thermoresponsive gel (TRG) combined with a double mutant of a bacterial heat-labile toxin (dmLT) for sublingual immunization with a trivalent inactivated poliovirus vaccine (IPV) in mice. This TRG delivery system, which changes from aqueous solution to viscous gel upon contact with the mucosa at body temperature, helps to retain the formulation at the site of delivery and has functional adjuvant activity from the inclusion of dmLT. IPV was administered to mice either sublingually in the TRG delivery system or intramuscularly in phosphate-buffered saline. We measured poliovirus type-specific serum neutralizing antibodies as well as polio-specific serum Ig and IgA antibodies in serum, saliva, and fecal samples using enzyme-linked immunosorbent assays. Mice receiving sublingual vaccination via the TRG delivery system produced both mucosal and serum antibodies, including IgA. Intramuscularly immunized animals produced only serum neutralizing and binding Ig but no detectable IgA. This study provides proof of concept for sublingual immunization using the TRG delivery system, comprising a thermoresponsive gel and dmLT adjuvant.

A role for IL-27 in limiting T regulatory cell populations.

IL-27 is a cytokine that regulates Th function during autoimmune and pathogen-induced immune responses. Although previous studies have shown that regulatory T cells (Tregs) express the IL-27R, and that IL-27 inhibits forkhead box P3 upregulation in vitro, little is known about how IL-27 influences Tregs in vivo. The studies presented in this article show that mice that overexpress IL-27 had decreased Treg frequencies and developed spontaneous inflammation. Although IL-27 did not cause mature Tregs to downregulate forkhead box P3, transgenic overexpression in vivo limited the size of a differentiating Treg population in a bone marrow chimera model, which correlated with reduced production of IL-2, a vital cytokine for Treg maintenance. These data identify an indirect role for IL-27 in shaping the Treg pool.

A role for IL-27p28 as an antagonist of gp130-mediated signaling.

The heterodimeric cytokine interleukin 27 (IL-27) signals through the IL-27Rα subunit of its receptor, combined with gp130, a common receptor chain used by several cytokines, including IL-6. Notably, the IL-27 subunits p28 (IL-27p28) and EBI3 are not always expressed together, which suggests that they may have unique functions. Here we show that IL-27p28, independently of EBI3, antagonized cytokine signaling through gp130 and IL-6-mediated production of IL-17 and IL-10. Similarly, the ability to generate antibody responses was dependent on the activity of gp130-signaling cytokines. Mice transgenic for expression of IL-27p28 showed a substantial defect in the formation of germinal centers and antibody production. Thus, IL-27p28, as a natural antagonist of gp130-mediated signaling, may be useful as a therapeutic for managing inflammation mediated by cytokines that signal through gp130.

Diversity of the CD8+ T-cell response to herpes simplex virus type 2 proteins among persons with genital herpes.

Cytolytic T cells play a major role in controlling herpes simplex virus type 2 (HSV-2) infections in humans. In an effort to more thoroughly evaluate the response to HSV-2 directly, ex vivo, we developed an enzyme-linked immunospot (ELISPOT) assay that utilized pools of overlapping synthetic peptides presented by autologous dendritic cells to purified CD8(+) T cells. Donor response rates to individual open reading frames (ORFs) ranged from fewer than 5% responding to as many as 70% responding, with the greatest frequency of responses (by ORF) being directed against UL39, UL25, UL27, ICP0, UL46, and UL47 in descending order of frequency. HSV-2-seropositive subjects responded to as few as 3 or as many as 46 of the 48 ORFs tested, with a median of 11 ORFs recognized. HLA-B*07 expression correlated with stronger responses overall that were directed primarily against UL49 and UL46. Cumulative precursor frequencies in the blood ranged from 500 to almost 6,000 HSV-2 spot-forming units/10(6) CD8(+) T cells. The magnitude and breadth of the response in the infected population were greater than previously appreciated. Whether this variability in the CD8(+) T-cell response within individuals is associated with the frequency of viral reactivation warrants further study.

Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects.

Human cytomegalovirus (HCMV) infections of immunocompetent hosts are characterized by a dynamic, life-long interaction in which host immune responses, particularly of T cells, restrain viral replication and prevent disease but do not eliminate the virus or preclude transmission. Because HCMV is among the largest and most complex of known viruses, the T cell resources committed to maintaining this balance have never been characterized completely. Here, using cytokine flow cytometry and 13,687 overlapping 15mer peptides comprising 213 HCMV open reading frames (ORFs), we found that 151 HCMV ORFs were immunogenic for CD4(+) and/or CD8(+) T cells, and that ORF immunogenicity was influenced only modestly by ORF expression kinetics and function. We further documented that total HCMV-specific T cell responses in seropositive subjects were enormous, comprising on average approximately 10% of both the CD4(+) and CD8(+) memory compartments in blood, whereas cross-reactive recognition of HCMV proteins in seronegative individuals was limited to CD8(+) T cells and was rare. These data provide the first glimpse of the total human T cell response to a complex infectious agent and will provide insight into the rules governing immunodominance and cross-reactivity in complex viral infections of humans.

Development of a therapeutic vaccine for HSV-2.

A therapeutic vaccine for genital herpes is clearly needed. Vaccines recently developed for HSV-2 in humans have been aimed at eliciting CD4+ T cell responses and neutralizing antibody responses to two HSV-2 glycoproteins (gB and gD). These vaccines have had no therapeutic effect against HSV-2 in human clinical trials. To enable development of an efficacious vaccine, Corixa Corporation has made a major effort to identify novel antigens that can be recognized by human HSV-2-specific CD8+ and CD4+ T cells. Corixa Corporation's proprietary adjuvants and delivery systems, when combined with appropriate antigens, may allow the development of an effective therapeutic vaccine for HSV-2.

Immunodominance among herpes simplex virus-specific CD8 T cells expressing a tissue-specific homing receptor.

The study of immunodominance within microbe-specific CD8 T cell responses has been challenging. We used a previously undescribed approach to create unbiased panels of CD8 cytotoxic T lymphocyte clones specific for herpes simplex virus type 2, a pathogen with a complex genome encoding at least 85 polypeptides. Circulating herpes simplex virus type 2-specific cells were enriched and cloned after sorting for expression of the skin homing-associated receptor, cutaneous lymphocyte-associated antigen, bypassing restimulation with antigen. The specificity of the resultant cytotoxic clones was determined. Clonal frequencies were compared with each other and with the total number of cytotoxic clones. For each subject within the homing receptor-positive compartment, the CD8 cytotoxic response was dominated by T cells specific for only a few peptides. Previously undescribed antigens and epitopes in viral tegument, capsid, or scaffold proteins were immunodominant in some subjects. Clone enumeration analyses were confirmed in some subjects with dominance studies by using herpes simplex mutants, vaccinia recombinants, and/or enzyme-linked immune spots. We conclude that among circulating cells expressing a homing-associated receptor, during chronic herpes type 2 infection, the CD8 T cell response becomes quite focused despite the presence of many potential antigenic peptides.

T cell immunity to herpes simplex viruses in seronegative subjects: silent infection or acquired immunity?

During the course of investigating T cell responses to HSV among volunteers entering trials of investigational genital herpes vaccines, 6 of the 24 immunocompetent subjects with no prior history of oral/labial or genital herpes possessed HSV-specific T cell immunity but, by multiple determinants of even the most sensitive serological assays, remained seronegative to HSV-1 and -2. Of these six immune seronegative (IS; HSV-seronegative with HSV-specific T cell responses) subjects, two had transient HSV-specific T cell responses, while four had CD4(+) and CD8(+) T cell responses directed at HSV that persisted for up to 4 years. CD4(+) T cell clones were isolated that recognized and had high binding affinities to epitopes in HSV-2 tegument proteins. All six IS subjects had potential sexual exposure to an HSV-2-infected sexual partner. Oral and genital mucosal secretions were sampled and tested for the presence of infectious HSV and HSV DNA. No evidence of HSV was detected in >1500 samples obtained from these IS subjects. The identification of persistent T cell responses to HSV in seronegative subjects is a novel finding in the herpesvirus field and suggests either undetected infection or acquired immunity in the absence of infection. Understanding the basis of these acquired immune responses may be critical in developing effective vaccines for genital herpes.