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Revisión This review article incorporates information from the 4th Global Meningococcal Initiative summit meeting. Since the introduction of stringent COVID-19 infection control and lockdown measures globally in 2020, there has been an impact on IMD prevalence, surveillance, and vaccination compliance. Incidence rates and associated mortality fell across various regions during 2020. A reduction in vaccine uptake during 2020 remains a concern globally. In addition, several Neisseria meningitidis clonal complexes, particularly CC4821 and CC11, continue to exhibit resistance to antibiotics, with resistance to ciprofloxacin or beta-lactams mainly linked to modifications of gyrA or penA alleles, respectively. Beta-lactamase acquisition was also reported through horizontal gene transfer (blaROB-1) involving other bacterial species. Despite the challenges over the past year, progress has also been made on meningococcal vaccine development, with several pentavalent (serogroups ABCWY and ACWYX) vaccines currently being studied in late-stage clinical trial programmes. Medical writing support was funded by Sanofi Pasteur. Sí

Pneumococcal conjugate vaccines (PCVs) have proven to be the best way to prevent severe childhood pneumococcal disease but until recently have been difficult for many countries to afford sustainabl...

Background Broadly protective pneumococcal vaccines that are affordable for low-resource countries are needed. Streptococcus pneumoniae whole cell vaccine (wSp) is an investigational vaccine that contains killed cells from a nonencapsulated strain of S. pneumoniae (SPn) with aluminum hydroxide adjuvant. Studies in mice demonstrated protection against nasopharyngeal carriage (T-cell-mediated) and invasive pneumococcal disease (antibody-mediated). The aim of this randomized, double-blind, placebo-controlled Phase 1 study was to assess safety, tolerability and immunogenicity of wSp in healthy adults. Methods Forty-two participants were randomized into 3 dose cohorts to receive 0.1, 0.3, or 0.6 mg of wSp or saline intramuscularly. Participants received a 3-dose vaccination schedule spaced by 4-week intervals. Postvaccination assessments included solicited reactogenicity events through day 7, blood chemistry and hematology assessments at day 7, and adverse events (AEs) through day 84. Participants were monitored for serum antibody and peripheral blood mononuclear cell cytokine responses to pneumococcal antigens. A 6-month telephone follow-up was completed to assess for any additional AEs. Results wSp was safe and well tolerated. Reactogenicity was acceptable and no untoward safety signals were observed. wSp elicited potentially clinically significant rises (defined arbitrarily as at least a 2-fold rise) in immunoglobulin G responses to multiple pneumococcal antigens, including pneumococcal surface protein A and pneumolysin. Functional antibody responses were observed with the highest dose of wSp (0.6 mg). Increases in T-cell cytokine responses, including interleukin 17A, were also seen among wSp vaccines. Conclusions wSp was safe and well tolerated in healthy US adults, eliciting pneumococcal antigen-specific antibody and T-cell cytokine responses.

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.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.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.).

Towards achieving the goal of eliminating epidemic outbreaks of meningococcal disease in the African meningitis belt, a pentavalent glycoconjugate vaccine (NmCV-5) has been developed to protect against Neisseria meningitidis serogroups A, C, Y, W and X. MenA and X polysaccharides are conjugated to tetanus toxoid (TT) while MenC, Y and W polysaccharides are conjugated to recombinant cross reactive material 197 (rCRM197), a non-toxic genetic variant of diphtheria toxin. This study describes quality control testing performed by the manufacturer, Serum Institute of India Private Limited (SIIPL), and the independent control laboratory of the U.K. (NIBSC) on seven clinical lots of the vaccine to ensure its potency, purity, safety and consistency of its manufacturing. In addition to monitoring upstream-manufactured components, samples of drug substance, final drug product and stability samples were evaluated. This paper focuses on the comparison of the vaccine’s critical quality attributes and reviews key indicators of its stability and immunogenicity. Comparable results were obtained by the two laboratories demonstrating sufficient levels of polysaccharide O-acetylation, consistency in size of the bulk conjugate molecules, integrity of the conjugated saccharides in the drug substance and drug product, and acceptable endotoxin content in the final drug product. The freeze-dried vaccine in 5-dose vials was stable based on molecular sizing and free saccharide assays. Lot-to-lot manufacturing consistency was also demonstrated in preclinical studies for polysaccharide-specific IgG and complement-dependent serum bactericidal activity for each serogroup. This study demonstrates the high quality and stability of NmCV-5, which is now undergoing Phase 3 clinical trials in Africa and India.

Despite advances in the development and introduction of vaccines against the major bacterial causes of meningitis, the disease and its long-term after-effects remain a problem globally. The Global Roadmap to Defeat Meningitis by 2030 aims to accelerate progress through visionary and strategic goals that place a major emphasis on preventing meningitis via vaccination. Global vaccination against Haemophilus influenzae type B (Hib) is the most advanced, such that successful and low-cost combination vaccines incorporating Hib are broadly available. More affordable pneumococcal conjugate vaccines are becoming increasingly available, although countries ineligible for donor support still face access challenges and global serotype coverage is incomplete with existing licensed vaccines. Meningococcal disease control in Africa has progressed with the successful deployment of a low-cost serogroup A conjugate vaccine, but other serogroups still cause outbreaks in regions of the world where broadly protective and affordable vaccines have not been introduced into routine immunization programs. Progress has lagged for prevention of neonatal meningitis and although maternal vaccination against the leading cause, group B streptococcus (GBS), has progressed into clinical trials, no GBS vaccine has thus far reached Phase 3 evaluation. This article examines current and future efforts to control meningitis through vaccination.

Objective To review and highlight significant advances made towards vaccine development and understanding of the immunology of otitis media (OM) since the 19th International Symposium on Recent Advances in Otitis Media (ISOM) in 2015, as well as identify future research directions and knowledge gaps. Data sources PubMed database, National Library of Medicine. Review methods Key topics were assigned to each panel member for detailed review. Draft reviews were collated, circulated, and thoroughly discussed when the panel met at the 20th ISOM in June 2019. The final manuscript was prepared with input from all panel members. Conclusions Since 2015 there have been a number of studies assessing the impact of licensed pneumococcal vaccines on OM. While these studies have confirmed that these vaccines are effective in preventing carriage and/or disease caused by vaccine serotypes, OM caused by non-vaccine serotype pneumococci and other otopathogens remains a significant health care burden globally. Development of multi-species vaccines is challenging but essential to reducing the global burden of OM. Influenza vaccination has been shown to prevent acute OM, and with novel vaccines against nontypeable Haemophilus influenzae (NTHi), Moraxella catarrhalis and Respiratory Syncytial Virus (RSV) in clinical trials, the potential to significantly prevent OM is within reach. Research into alternative vaccine delivery strategies has demonstrated the power of maternal and mucosal vaccination for OM prevention. Future OM vaccine trials must include molecular diagnostics of middle ear effusion, for detection of viruses and bacteria that are persisting in biofilms and to enable accurate assessment of vaccine impact on OM etiology. Understanding population differences in natural and vaccine-induced immune responses to otopathogens is also important for development of the most effective OM vaccines. Improved understanding of the interaction between otopathogens will also advance development of effective therapies and encourage the assessment of the indirect benefits of vaccination. Implications for practice While NTHi and M. catarrhalis are the predominant otopathogens, funding opportunities to drive vaccine development for these species are limited due to a focus on prevention of childhood mortality rather than morbidity. Delivery of a comprehensive report on the high financial and social costs of OM, including the potential for OM vaccines to reduce antibiotic use and subsequent development of antimicrobial resistance (AMR), would likely assist in engaging stakeholders to recognize the value of prevention of OM and increase support for efforts on OM vaccine development. Vaccine trials with OM prevention as a clinical end-point are challenging, however a focus on developing assays that measure functional correlates of protection would facilitate OM vaccine development.

Background A more affordable pneumococcal conjugate vaccine (PCV) that provides comparable protection to current PCVs is needed to ensure sustainable access in resource-limited settings. Serum Institute of India Pvt. Ltd.’s PCV candidate (SIIPL-PCV) has the potential to meet this need as manufacturing efficiency has been optimized and the vaccine targets the most prevalent disease-causing serotypes in Africa and Asia. We report SIIPL-PCV’s safety, tolerability, and immunogenicity in adults, toddlers, and infants in The Gambia. Methods This phase 1/2, randomized, double-blind trial sequentially enrolled 34 PCV-naive adults (18–40 years old), 112 PCV (Prevenar 13® [PCV13])-primed toddlers (12–15 months old), and 200 PCV-naive infants (6–8 weeks old), who were randomized (1:1) to receive SIIPL-PCV or a licensed comparator vaccine. Infants received three-doses of SIIPL-PCV or PCV13 at 6, 10, and 14 weeks of age co-administered with routine Expanded Program on Immunization (EPI) vaccines. Reactogenicity was solicited through seven-days post-vaccination; unsolicited adverse events (AEs) were assessed throughout the study. The safety and immunogenicity of a matching booster at 10–14 months of age were evaluated in a subset of 96 infants. Immune responses were evaluated post-primary and pre- and post-booster vaccinations. Results Reactogenicity was primarily mild-to-moderate in severity. In infants, the most common solicited reactions were injection-site tenderness and fever, with no meaningful treatment-group differences. There were no serious or severe vaccine-related AEs and no meaningful trends in SAEs, vaccine-related AEs, or overall AEs. Infant post-primary seroresponse rates (IgG level ≥ 0.35 µg/mL) were ≥89% for all serotypes except 6A (79%) in the SIIPL-PCV group. IgG GMCs were >1 µg/mL for all serotypes in both SIIPL-PCV and PCV13 groups. Post-booster GMCs were comparable between groups. Conclusion SIIPL-PCV was well-tolerated, had an acceptable safety profile, and was immunogenic for all vaccine serotypes. Results support the evaluation of SIIPL-PCV in a phase 3 non-inferiority trial. Clinicaltrials.gov: NCT02308540 .

Background Two conserved pneumococcal proteins, pneumolysin toxoid (dPly) and pneumococcal histidine triad protein D (PhtD), combined with 10 polysaccharide conjugates from the pneumococcal non-typeable Haemophilus influenzae protein D-conjugate vaccine (PHiD-CV) in two investigational pneumococcal vaccine (PHiD-CV/dPly/PhtD) formulations were immunogenic and well-tolerated when administered to Gambian children. Here, we report immunogenicity of the polysaccharide conjugates, and immunogenicity and reactogenicity of co-administered routine vaccines. Methods In this phase II, controlled, observer-blind, single-centre study, healthy infants aged 8–10 weeks were randomised (1:1:1:1:1:1) to six groups. Four groups received 3+0 schedule (2-3-4 months [M]) of PHiD-CV/dPly/PhtD (10 or 30 µg of each protein), PHiD-CV, or 13-valent pneumococcal conjugate vaccine; and two groups received 2+1 schedule (2-4-9 M) of PHiD-CV/dPly/PhtD (30 µg of each protein) or PHiD-CV. All infants received diphtheria-tetanus-whole cell pertussis-hepatitis B-Haemophilus influenzae type b (DTPw-HBV/Hib) and oral trivalent polio vaccines (OPV) at 2-3-4 M, and measles, yellow fever, and OPV vaccines at 9 M. We evaluated immune responses at 2-5-9-12 M; and reactogenicity 0–3 days post-vaccination. Results 1200 infants were enrolled between June 2011 and May 2012; 1152 completed the study. 1 M post-primary vaccination, for each PHiD-CV serotype except 6B and 23F, ≥97.4% (3+0 schedule) and ≥96.4% (2+1 schedule) of infants had antibody concentrations ≥0.2 μg/mL. Immune responses were comparable between groups within the same vaccination schedules. Observed antibody geometric mean concentrations (GMCs) increased by 1 M post-primary vaccination compared to pre-vaccination. In the following months, GMCs and opsonophagocytic activity titres waned, with an increase post-booster for the 2+1 schedule. Immune responses to protein D and, DTPw-HBV/Hib, OPV, measles, and yellow fever vaccines were not altered by co-administration with pneumococcal proteins. Reactogenicity of co-administered vaccines was comparable between groups and did not raise concerns. Conclusion Immune responses to the 10 PHiD-CV polysaccharide conjugates and co-administered vaccines were not altered by addition of dPly and PhtD. ClinicalTrials.gov identifier NCT01262872.

Pneumococcal conjugate vaccines (PCVs) have been successful in preventing invasive pneumococcal disease but effectiveness has been challenged by replacement of vaccine serotypes with non-vaccine serotypes. Vaccines targeting common pneumococcal protein(s) found in most/all pneumococci may overcome this limitation. This phase II study assessed safety and immunogenicity of a new protein-based pneumococcal vaccine containing polysaccharide conjugates of 10 pneumococcal serotypes combined with pneumolysin toxoid(dPly) and pneumococcal histidine triad protein D(PhtD) (PHiD-CV/dPly/PhtD-30) in African children. 120 Gambian children (2-4 years, not previously vaccinated against Streptococcus pneumoniae) randomized (1:1) received a single dose of PHiD-CV/dPly/PhtD-30 or PCV13. Adverse events occurring over 4 d post-vaccination were reported, and blood samples obtained pre- and 1-month post-vaccination. Serious adverse events were reported for 6 months post-vaccination. Solicited local and systemic adverse events were reported at similar frequency in each group. One child (PHiD-CV/dPly/PhtD-30 group) reported a grade 3 local reaction to vaccination. Haematological and biochemical parameters seemed similar pre- and 1-month post-vaccination in each group. High pre-vaccination Ply and PhtD antibody concentrations were observed in each group, but only increased in PHiD-CV/dPly/PhtD-30 vaccinees one month post-vaccination. One month post-vaccination, for each vaccine serotype ≥96.2% of PHiD-CV/dPly/PhtD-30 vaccinees had serotype-specific polysaccharide antibody concentrations ≥0.20µg/mL except serotypes 6B (80.8%) and 23F (65.4%), and ≥94.1% had OPA titres of ≥8 except serotypes 1 (51.9%), 5 (38.5%) and 6B (78.0%), within ranges seen in PCV13-vaccinated children. A single dose of PHiD-CV/dPly/PhtD-30 vaccine, administered to Gambian children aged 2-4 y not previously vaccinated with a pneumococcal vaccine, was well-tolerated and immunogenic.

Summary 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.

Highlights • Pneumococcal conjugate vaccines (PCVs) have significantly reduced invasive pneumococcal disease (IPD) and pneumococcal pneumonia. • PCV impact on IPD is blunted by serotype replacement. • Pneumococcal protein vaccines may provide broad, serotype independent protection., Pneumococcal disease is a major cause of morbidity and mortality in young children, particularly in the developing world. Vaccines are a critical strategy for protecting children from pneumococcal disease and licensed pneumococcal conjugate vaccines (PCVs) are having a significant impact on invasive pneumococcal disease and pneumococcal pneumonia throughout the world. Currently available PCVs do not, however, cover all pneumococcal serotypes and are complicated and relatively expensive to manufacture. While new PCV development is focused on either higher valency or more inherent affordability for developing countries, new vaccines are needed that offer serotype-independent protection. Vaccines containing proteins that are common to all pneumococcal serotypes could provide broad protection to children worldwide. Protein subunit and whole cell vaccines have advanced into Phase 1 and 2 clinical trials but face considerable challenges before they can become licensed and widely distributed.

Background Conserved pneumococcal proteins are potential candidates for inclusion in vaccines against pneumococcal diseases. In the first part of a two-part study, an investigational vaccine (PHiD-CV/dPly/PhtD-30) containing 10 pneumococcal serotype-specific polysaccharide conjugates (10VT) combined with pneumolysin toxoid and pneumococcal histidine triad protein D (30 μg each) was well tolerated by Gambian children. Part two, presented here, assessed the efficacy of two PHiD-CV/dPly/PhtD formulations against pneumococcal nasopharyngeal carriage (NPC) prevalence in infants. Methods In this phase 2, randomized, controlled, observer-blind trial, healthy infants aged 8–10 weeks, recruited from a peri-urban health center, were randomized (1:1:1:1:1:1) into six groups. Four groups received PHiD-CV/dPly/PhtD (10 or 30 μg of each protein), PHiD-CV, or 13-valent pneumococcal conjugate vaccine at ages 2–3–4 months (3 + 0 infant schedule) and two groups PHiD-CV/dPly/PhtD-30 or PHiD-CV at 2–4–9 months (2 + 1 infant schedule). The primary objective was impact on non-10VT NPC at ages 5–9–12 months. Secondary objectives included confirmatory analysis of protein dose superiority and safety/reactogenicity. Impact on pneumococcal NPC acquisition, bacterial load, and ply and phtD gene sequencing were explored. Results 1200 infants were enrolled between June 2011 and May 2012. Prevalences of pneumococcal (60–67%) and non-10VT (55–61%) NPC were high at baseline. Across all post-vaccination time points, efficacy of PHiD-CV/dPly/PhtD-10 and PHiD-CV/dPly/PhtD-30 against non-10VT NPC (3 + 0 schedule) was 1.1% (95% CI −21.5, 19.5) and 2.1% (−20.3, 20.3), respectively; efficacy of PHiD-CV/dPly/PhtD-30 (2 + 1 schedule) was 0.5% (−22.1, 18.9) versus PHiD-CV. No differences were observed in pneumococcal NPC acquisition, clearance, or bacterial load. Both protein-based vaccines elicited immune responses to pneumococcal proteins. Conclusions In this high carriage prevalence setting, inclusion of pneumococcal proteins in the PHiD-CV/dPly/PhtD investigational vaccine had no impact on pneumococcal NPC in infants, regardless of protein dose or schedule. Future evaluations will assess its impact against pneumococcal disease endpoints. Funding: PATH, GlaxoSmithKline Biologicals SA. ClinicalTrials.gov identifier NCT01262872 .

To update progress on the effectiveness of vaccine for prevention of acute otitis media (AOM) and identification of promising candidate antigens against Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis.Literature searches were performed in OvidSP and PubMed restricted to articles published between June 2007 and September 2011. Search terms included otitis media, vaccines, vaccine antigens, and each of the otitis pathogens and candidate antigens identified in the ninth conference report.The current report provides further evidence for the effectiveness of pneumococcal conjugate vaccines (PCVs) in the prevention of otitis media. Observational studies demonstrate a greater decline in AOM episodes than reported in clinical efficacy trials. Unmet challenges include extending protection to additional serotypes and additional pathogens, the need to prevent early episodes, the development of correlates of protection for protein antigens, and the need to define where an otitis media vaccine strategy fits with priorities for child health.Acute otitis media continues to be a burden on children and families, especially those who suffer from frequent recurrences. The 7-valent PCV (PCV7) has reduced the burden of disease as well as shifted the pneumococcal serotypes and the distribution of otopathogens currently reported in children with AOM. Antibiotic resistance remains an ongoing challenge. Multiple candidate antigens have demonstrated the necessary requirements of conservation, surface exposure, immunogenicity, and protection in animal models. Further research on the role of each antigen in pathogenesis, in the development of correlates of protection in animal models, and in new adjuvants to elicit responses in the youngest infants is likely to be productive and permit more antigens to move into human clinical trials.

Globally, group B Streptococcus (GBS) remains a leading cause of sepsis and meningitis in infants in the first 90 days of life. Intrapartum antibiotic prophylaxis (IAP) for women at increased risk of transmitting GBS to their newborns has been effective in reducing part, but not all, of the GBS disease burden in many high income countries (HICs). In low- and middle-income countries (LMICs), IAP use is low. Immunization of pregnant women with a GBS vaccine represents an alternative strategy to protecting newborns and young infants, through transplacental antibody transfer and potentially by reducing new vaginal colonization. This vaccination strategy was first suggested in the 1970s and several potential GBS vaccines have completed phase I/II clinical trials. During the 2015 WHO Product Development for Vaccines Advisory Committee meeting, GBS was identified as a high priority for the development of a vaccine for maternal immunization because of the major public health burden posed by GBS in LMICs, and the high technical feasibility for successful development. Following this meeting, the first WHO technical consultation on GBS vaccines was held on the 27th and 28th of April 2016, to consider development pathways for such vaccines, focused on their potential role in reducing newborn and young infant deaths and possibly stillbirths in LMICs. Discussion topics included: (1) pathophysiology of disease; (2) current gaps in the knowledge of global disease burden and serotype distribution; (3) vaccine candidates under development; (4) design considerations for phase III trials; and (5) pathways to licensure, policy recommendations and use. Efforts to address gaps identified in each of these areas are needed to establish the public health need for, the development and deployment of, efficacious GBS vaccines. In particular, more work is required to understand the global disease burden of GBS-associated stillbirths, and to develop quality-assured standardized antibody assays to identify correlates of protection.

Background. Pneumococcus, meningococcus, and Haemophilus influenzae cause a similar spectrum of infections in the ear, lung, blood, and brain. They share cross-reactive antigens that bind to the laminin receptor of the blood-brain barrier as a molecular basis for neurotropism, and this step in pathogenesis was addressed in vaccine design. Methods. Biologically active peptides derived from choline-binding protein A (CbpA) of pneumococcus were identified and then genetically fused to L460D pneumolysoid. The fusion construct was tested for vaccine efficacy in mouse models of nasopharyngeal carriage, otitis media, pneumonia, sepsis, and meningitis. Results. The CbpA peptide–L460D pneumolysoid fusion protein was more broadly immunogenic than pneumolysoid alone, and antibodies were active in vitro against Streptococcus pneumoniae, Neisseria meningitidis, and H. influenzae. Passive and active immunization protected mice from pneumococcal carriage, otitis media, pneumonia, bacteremia, meningitis, and meningococcal sepsis. Conclusions. The CbpA peptide–L460D pneumolysoid fusion protein was broadly protective against pneumococcal infection, with the potential for additional protection against other meningeal pathogens.

In view of the increasing licensure and use of pneumococcal conjugate vaccines, the relatively high cost, and growing issues with serotype emergence, there is a need to re-evaluate the role of pneumococcal protein vaccines (PPVs) and pathways to their licensure. This paper summarizes the discussion and viewpoints from an expert meeting regarding the development of PPVs. A wide spectrum of pneumococcal vaccine researchers, developers, and regulators met to review the state of PPVs, identify research and development needs, and provide consensus opinions to support the introduction of new PPVs where possible. They also discussed clinical and regulatory aspects pertinent to these vaccines and generated a series of recommendations for moving the field forward.

In view of the increasing use of pneumococcal vaccines, especially in the developing world, there is a need for appropriate diagnostics to understand the aetiology of pneumonia, to define the burden of pneumococcal disease, and to monitor vaccine efficacy and effectiveness. This article summarizes a meeting on the diagnosis, detection and serotyping of pneumococcal disease organized by PATH and Fondation Mérieux (18–20 October 2009, Fondation Mérieux Conference Centre, Les Pensières, France). Workers and experts met to discuss the gaps in the microbiology-based diagnosis of Streptococcus pneumoniae disease, with special emphasis on pneumonia. The meeting was designed to evaluate the state of the art of pneumococcal diagnostics and serotyping methodologies, identify research and development needs, and propose new guidelines to public health authorities to support the introduction of vaccines. Regarding detection, the main recommendations were to encourage chest X-rays and antigen detection in urine. Large-scale studies are needed to evaluate the diagnostic utility of test algorithms that associate chest X-rays, antigen detection in urine, S. pneumoniae quantitative PCR in nasopharyngeal aspirates and sputum, and C-reactive protein or procalcitonin measurement in blood. Efforts should be focused on proteomics to identify pneumococcus-specific antigens in urine or host markers in blood expressed during pneumonia. It was recommended to develop S. pneumoniae typing capacities, to understand the epidemiology of pneumococcal disease, and to evaluate vaccine effectiveness. Simple and effective approaches are encouraged, and new technologies based on beads, microarrays or deep sequencing should be developed to determine, in a single test capsular serotype, resistance profile and genotype.

SummaryStreptococcus pneumoniae is a leading cause of mortality in young children. While successful conjugate polysaccharide vaccines exist, a less expensive serotype-independent protein-based pneumococcal vaccine offers a major advancement for preventing life-threatening pneumococcal infections, particularly in developing nations. IL-17A-secreting CD4+ T cells (TH17) mediate resistance to mucosal colonization by multiple pathogens including S. pneumoniae. Screening an expression library containing >96% of predicted pneumococcal proteins, we identified antigens recognized by TH17 cells from mice immune to pneumococcal colonization. The identified antigens also elicited IL-17A secretion from colonized mouse splenocytes and human PBMCs suggesting that similar responses are primed during natural exposure. Immunization of two mouse strains with identified antigens provided protection from pneumococcal colonization that was significantly diminished in animals treated with blocking CD4 or IL-17A antibodies. This work demonstrates the potential of proteomic screening approaches to identify specific antigens for the design of subunit vaccines against mucosal pathogens via harnessingTH17-mediated immunity.

Mucosal immunization with a killed whole-cell pneumococcal vaccine, given with enterotoxin-related adjuvants, has been shown to confer multi-serotype protection against colonization of the nasopharynx and middle ear in mice. However, because novel mucosal immunization strategies may be difficult to implement, here we evaluated subcutaneous injection. Strain RM200 was engineered to be capsule-negative, autolysin-negative, and to express a non-toxic mutant pneumolysoid. Liter-scale and 60-l Good Manufacturing Practice (GMP) cultures were grown in bovine-free soy-based medium, killed with chloroform or beta-propiolactone, and injected into C57Bl/6 mice without or with aluminum adjuvant. The adjuvant Al(OH)(3) strongly increased responses, particularly if pre-treated with phosphate. Protection was found in several tested model infections: nasal colonization with a serotype 6B strain and fatal aspiration-sepsis with strains of serotype 3 and 5. Protection against colonization was mechanistically dependent on the presence of CD4+ T cells at the time of challenge; in contrast, in the type 3 aspiration-sepsis model, CD4+ T cells were not required for protection at the time of challenge, suggesting that antibody alone was sufficient to protect against death in this model. Rabbits receiving sequential intramuscular injections in a pilot toxicity study displayed local reactogenicity at injection sites but no clinical signs. The rabbit antiserum thus produced was active in an in vitro phagocytic killing assay and passively protected mice in the type 3 aspiration-sepsis model. Approval is being sought for human trials of this vaccine.

Streptococcus pneumoniae kills nearly 1 million children before their fifth birthdays each year, mostly in the developing world. Current pneumococcal vaccines provide effective, serotype-specific protection, yet no licensed vaccine today offers broad coverage against all of S. pneumoniae’s more than 90 serotypes. Although low-income countries are beginning to gain access to these lifesaving vaccines via assistance from global health partnerships, such vaccines are otherwise unaffordable for poorer nations due to the relative complexity and the high costs associated with their development and production. Over the long term, new vaccines are needed that can maximize protection in the developing world and that even the poorest countries can sustainably afford without assistance. To this end, PATH’s pneumococcal vaccine project is working with public- and private-sector partners to develop safe, affordable and effective vaccines against S. pneumoniae tailored to meet the needs of infants and children in the developing world.

We previously reported that ethanol-killed cells of a noncapsulated strain of Streptococcus pneumoniae , given intranasally with cholera toxin as an adjuvant, protect rats against pneumonia and mice against colonization of the nasopharynx and middle ear by capsulated pneumococci of various serotypes. The acceleration of pneumococcal clearance from the nasopharynx in mice is CD4 + T cell-dependent and interleukin 17A (IL-17A) mediated and can be antibody independent. Here, anticipating human studies, we have demonstrated protection with a new vaccine strain expressing a nonhemolytic derivative of pneumolysin and grown in bovine-free culture medium. Killing the cells with chloroform, trichloroethylene, or beta-propiolactone—all used without postinactivation washing—produced more-potent immunogens than ethanol, and retention of soluble components released from the cells contributed to protection. Two sequential intranasal administrations of as little as 1 μg of protein (total of cellular and soluble combined) protected mice against nasopharyngeal challenge with pneumococci. Nontoxic single and double mutants of Escherichia coli heat-labile toxin were effective as mucosal adjuvants. Protection was induced by the sublingual and buccal routes, albeit requiring larger doses than when given intranasally. Protection was likewise induced transdermally with sonicates of the killed-cell preparation. Thus, this whole-cell antigen can be made and administered in a variety of ways to suit the manufacturer and the vaccination program and is potentially a solution to the need for a low-cost vaccine to reduce the burden of childhood pneumococcal disease in low-income countries.

Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection worldwide, with over 91 million cases estimated annually. An effective subunit vaccine against Chlamydia may require a multivalent subunit cocktail of antigens in a single formulation for broad coverage of a heterogeneous major histocompatibility complex population. Herein, we describe the identification of novel C. trachomatis antigens by CD4+ and CD8+ T-cell expression cloning, serological expression cloning, and an in silico analysis of the C. trachomatis genome. These antigens elicited human CD4+ T-cell responses, and a subset proved to be immunogenic and protective when administered as immunoprophylactic vaccines against C. trachomatis challenge. Candidate vaccines consisting of the prioritized C. trachomatis antigens adjuvanted in a GlaxoSmithKline proprietary AS01B adjuvant were prioritized based on induction of solid protection against challenge in C57BL/6 and BALB/c mice with C. trachomatis. Some of the vaccines prevented bacterial shedding and colonization of the upper genital tract to varying degrees by mechanisms that may include CD4+ T cells.

The development of a vaccine for tuberculosis requires a combination of antigens and adjuvants capable of inducing appropriate and long-lasting T cell immunity. We evaluated Mtb72F formulated in AS02A in the cynomolgus monkey model. The vaccine was immunogenic and caused no adverse reactions. When monkeys were immunized with bacillus Calmette–Guérin (BCG) and then boosted with Mtb72F in AS02A, protection superior to that afforded by using BCG alone was achieved, as measured by clinical parameters, pathology, and survival. We observed long-term survival and evidence of reversal of disease progression in monkeys immunized with the prime-boost regimen. Antigen-specific responses from protected monkeys receiving BCG and Mtb72F/AS02A had a distinctive cytokine profile characterized by an increased ratio between 3 Th1 cytokines, IFN-γ, TNF, and IL-2 and an innate cytokine, IL-6. To our knowledge, this is an initial report of a vaccine capable of inducing long-term protection against tuberculosis in a nonhuman primate model, as determined by protection against severe disease and death, and by other clinical and histopathological parameters.

Immunization with the pneumococcal proteins pneumolysin (Ply), choline binding protein A (CbpA), or pneumococcal surface protein A (PspA) elicits protective responses against invasive pneumococcal disease in animal models. In this study, we used different mouse models to test the efficacy of a variety of multivalent protein-based vaccines that comprised various combinations of full-length or peptide regions of the immunogens Ply, CbpA, or PspA: Ply toxoid with the L460D substitution (referred to herein as L460D); L460D fused with protective peptide epitopes from CbpA (YPT-L460D-NEEK [YLN]); L460D fused with the CD2 peptide containing the proline-rich region (PRR) of PspA (CD2-L460D); a combination of L460D and H70 (L460D+H70), a slightly larger PspA-derived peptide containing the PRR and the SM1 region; H70+YLN; and other combinations. Each mouse was immunized either intraperitoneally (i.p.) or subcutaneously (s.c.) with three doses (at 2-week intervals) of the various antigen combinations in alum adjuvant and then challenged in mouse models featuring different infection routes with multiple Streptococcus pneumoniae strains. In the i.p. infection sepsis model, H70+YLN consistently provided significant protection against three different challenge strains (serotypes 1, 2, and 6A); the CD2+YLN and H70+L460D combinations also elicited significant protection. Protection against intravenous (i.v.) sepsis (type 3 and 6A challenge strains) was largely dependent on PspA-derived antigen components, and the most protection was elicited by H70 with or without L460D or YLN. In a type 4 intratracheal (i.t.) challenge model that results in progression to meningitis, antigen combinations that contained YLN elicited the strongest protection. Thus, the trivalent antigen combination of H70+YLN elicited the strongest and broadest protection in diverse pneumococcal challenge models.

Monophosphoryl lipid A (MPL®) is a potent vaccine adjuvant derived from Salmonella minnesota that was recently licensed in Europe as a component of an improved vaccine for hepatitis B (Fendrix®). MPL, like lipopolysaccharide from which it is derived, signals via the TLR4/MD-2 complex. We have produced a series of synthetic Toll-like receptor 4 (TLR4) agonists that are based upon the structure of the major hexa-acylated congener contained within MPL. These TLR4 agonists, termed the aminoalkyl glucosaminide phosphates (AGPs), stimulate the production of various cytokines by human peripheral blood mononuclear cells in vitro and up-regulate cell surface markers on monocytes, NK cells and B cells. In addition, AGPs provide non-specific resistance to challenge with viral and bacterial pathogens when administered to the upper airways of mice. Structure—activity relationship studies have shown that the activation of innate immune effectors by AGPs depends primarily on the length of the secondary acyl chains and the nature of the functional group attached to the aglycon component. Moreover, AGPs can act as potent adjuvants for mucosal administration of vaccine antigens, enhancing both antigen-specific antibody and cell-mediated immune responses. Thus, by combining the adjuvant and non-specific resistance induction properties of AGPs it may be possible to generate mucosal vaccines that provide innate protection immediately following administration together with long-term acquired immunity.

The development of novel vaccine strategies supplementingMycobacterium bovisBCG (BCG) constitutes an urgent research challenge. To identify potential subunit vaccine candidates, we have tested a series of eight recently identifiedMycobacterium tuberculosisantigens inM. bovis-infected and BCG-vaccinated cattle. These antigens were characterized on the basis of their ability to induce in vitro gamma interferon responses in infected or BCG-vaccinated calves. We were able to establish a hierarchy of these antigens based on how frequently they were recognized in both groups of animals. In particular, we were able to prioritize frequently recognized proteins like Rv0287, Rv1174, and Rv1196 for future evaluation as subunit vaccines to be used in BCG-protein heterologous prime-boost vaccination scenarios. In addition, the antigen most dominantly recognized inM. bovis-infected cattle in this study, Rv3616c, was significantly less frequently recognized by BCG vaccinees and could be a target to improve BCG, for example, by increasing its secretion, in a recombinant BCG vaccine.

CD8+ T cells play an important role in the host response to infection with Mycobacterium tuberculosis (Mtb). Mtb resides in an arrested phagosome that is phenotypically similar to an early endosome. The mechanisms by which Mtb-derived Ags gain access to the HLA-I-processing pathway are incompletely characterized. Studies with CD8+ T cell lines have suggested that Mtb Ags gain access to the HLA-I pathway in an alternate vacuolar pathway that is both brefeldin A (BFA) and TAP independent. To define the requirements of entry of Ag into the HLA-I pathway, we have used human CD8+ T cell clones specific for the secreted Mtb Ag CFP10. Human monocyte-derived dendritic cells were pulsed with CFP10 expressed in a recombinant adenovirus, surface adsorbed to microspheres, or in its native form by Mtb. When delivered by adenovirus, processing and presentation of CFP10 were blocked by both BFA and the proteasomal blocker lactacystin. In contrast, processing of CFP10 adsorbed to the surface of microspheres was not affected by either of these Ag-processing inhibitors. BFA, lactacystin, and TAP inhibition blocked the recognition of Mtb-infected dendritic cells, suggesting that processing was via a cytosolic pathway for this secreted protein Ag. We conclude that secreted proteins from Mtb can be processed in a BFA- and proteasome-dependent manner, consistent with egress of Ag into the cytosol and subsequent loading of proteasomally derived peptides.

MTB41 is a Mycobacterium antigen that is recognized by CD4+ T cells early after experimental infection of mice with Mycobacterium tuberculosis and by PBMC from healthy PPD positive individuals. Immunization of mice with plasmid DNA encoding the MTB41 gene sequence results in the development of antigen-specific CD4+ and CD8+ T cells, and protection against challenge with virulent M. tuberculosis. In the present studies, in contrast to DNA immunization, we show, that a strong MTB41-specific CD4+ T cell response, but no MHC class I restricted cytotoxic T lymphocyte (CTL) activity is detected in the spleen cells of infected mice. Therefore, this data suggests that the induction of CD8+ T cell response to MTB41 epitopes by DNA immunization may not be relevant to protection because these epitopes are not recognized during the infectious process. We also compared the repertoire of rMTB41 epitope recognition by CD4+ T cells of M. tuberculosis-infected mice with the recognition repertoire of mice immunized with the recombinant rMTB41 protein. Both regimens of sensitization lead to the recognition of the same molecular epitope. Coincidentally, immunization with the soluble recombinant protein plus adjuvant, a regimen known to generate primarily CD4+ T cells, resulted in induction of protection comparable to BCG in two well-established animal models of tuberculosis (mice and guinea pigs).

Chlamydia trachomatis is an obligate intracellular gram-negative bacterium responsible for a wide spectrum of diseases in humans. Both genital and ocular C. trachomatis infections are associated with tissue inflammation and pathology. Dendritic cells (DC) play an important role in both innate and adaptive immune responses to microbial pathogens and are a source of inflammatory cytokines. To determine the potential contribution of DC to the inflammatory process, human DC were infected with C. trachomatis serovar E or L2. Both C. trachomatis serovars were found to infect and replicate in DC. Upon infection, DC up-regulated the expression of costimulatory (B7-1) and cell adhesion (ICAM-1) molecules. Furthermore, chlamydial infection induced the secretion of interleukin-1β (IL-1β), IL-6, IL-8, IL-12p70, IL-18, and tumor necrosis factor alpha (TNF-α). The mechanisms involved in Chlamydia -induced IL-1β and IL-18 secretion differed from those of the other cytokines. Chlamydia -induced IL-1β and IL-18 secretion required infection with viable bacteria and was associated with the Chlamydia -induced activation of caspase-1 in infected host cells. In contrast, TNF-α and IL-6 secretion did not require that the Chlamydia be viable, suggesting that there are at least two mechanisms involved in the Chlamydia -induced cytokine secretion in DC. Interestingly, an antibody to Toll-like receptor 4 inhibited Chlamydia -induced IL-1β, IL-6, and TNF-α secretion. The data herein demonstrate that DC can be infected by human C. trachomatis serovars and that chlamydial components regulate the secretion of various cytokines in DC. Collectively, these data suggest that DC play a role in the inflammatory processes caused by chlamydial infections.

The intracellular bacterial pathogen Chlamydia is sequestered from the host cell cytoplasm by remaining within an inclusion body during its replication cycle. Nevertheless, CD8+ T cells recognizing Chlamydia Ags in the context of MHC class I molecules are primed during infection. We have recently described derivation of Chlamydia-specific human CD8+ T cells by using infected dendritic cells as a surrogate system to reflect Chlamydia-specific CD8+ T cell responses in vivo. These CD8+ T cell clones recognize chlamydial Ags processed via the conventional class Ia processing pathway, as assessed by treatment of infected APC with lactacystin and brefeldin A, suggesting that the Ags are translocated from the chlamydial inclusion into the host cell cytosol. In this study, outer membrane protein 2 (OmcB) was identified as the Ag recognized by one of these Chlamydia-specific human CD8+ T cells, and we defined the HLA*A0101-restricted epitope from this Ag. CD8+ T cell responses to this epitope were present at high frequencies in the peripheral blood of both of two HLA*A0101 donors tested. In vitro chlamydial growth was completely inhibited by the OmcB-specific CD8+ T cell clone independently of lytic mechanisms. OmcB is a 60-kDa protein that has been postulated to be associated with the Chlamydia outer membrane complex. The subcellular localization of OmcB to the cytosol of infected cells, as determined by conventional MHC class I Ag processing and presentation, suggests the possibility of an additional, cytosolic-associated function for this protein.

A tuberculosis vaccine candidate consisting of a 72-kDa polyprotein or fusion protein based upon the Mtb32 and Mtb39 antigens of Mycobacterium tuberculosis and designated Mtb72F was tested for its protective capacity as a potential adjunct to the Mycobacterium bovis BCG vaccine in the mouse and guinea pig models of this disease. Formulation of recombinant Mtb72F (rMtb72F) in an AS02A adjuvant enhanced the Th1 response to BCG in mice but did not further reduce the bacterial load in the lungs after aerosol challenge infection. In the more stringent guinea pig disease model, rMtb72F delivered by coadministration with BCG vaccination significantly improved the survival of these animals compared to BCG alone, with some animals still alive and healthy in their appearance at >100 weeks post-aerosol challenge. A similar trend was observed with guinea pigs in which BCG vaccination was boosted by DNA vaccination, although this increase was not statistically significant due to excellent protection conferred by BCG alone. Histological examination of the lungs of test animals indicated that while BCG controls eventually died from overwhelming lung consolidation, the majority of guinea pigs receiving BCG mixed with rMtb72F or boosted twice with Mtb72F DNA had mostly clear lungs with minimal granulomatous lesions. Lesions were still prominent in guinea pigs receiving BCG and the Mtb72F DNA boost, but there was considerable evidence of lesion healing and airway remodeling and reestablishment. These data support the hypothesis that the coadministration or boosting of BCG vaccination with Mtb72F may limit the lung consolidation seen with BCG alone and may promote lesion resolution and healing. Collectively, these data suggest that enhancing BCG is a valid vaccination strategy for tuberculosis that is worthy of clinical evaluation.

Recombinant immunodominant mycobacterial antigens are needed for the development of new vaccines and immunodiagnostic tools for use against tuberculosis. Ubiquitous exposure to mycobacteria in tropical countries could influence vaccine-induced immunity and the specificity of tuberculosis immunodiagnosis. For this study conducted in The Gambia, cellular immune responses to recombinant mycobacterial antigens were characterized inMycobacterium bovisBCG-vaccinated and nonvaccinated infants, adult community controls, household contacts, health care workers, and tuberculosis patients. Neonatal BCG vaccination induced gamma interferon (IFN-γ) responses to Mtb8.4, Mtb32-C, Mtb39A, Mtb9.9A, and Mtb32-N, but not CFP-10 (Mtb11) and α-crystallin (Mtb16). Exposure toMycobacterium tuberculosisin household contacts and health care workers was associated with high responses to CFP-10 and α-crystallin. Generally, low IFN-γ responses were found in tuberculosis patients. These results suggest that Mtb8.4, Mtb32-C, Mtb39A, Mtb9.9A, and Mtb32-N may be used in a subunit vaccine to boost BCG-induced immunity. While CFP-10 and α-crystallin are promising candidates for the immunodiagnosis ofM. tuberculosisinfection or for vaccine use, disease-associated immunosuppression may prevent IFN-γ immunodiagnosis of more advanced tuberculosis.

During its developmental cycle, the intracellular bacterial pathogen Chlamydia trachomatis remains confined within a protective vacuole known as an inclusion. Nevertheless, CD8+ T cells that recognize Chlamydia Ags in the context of MHC class I molecules are primed during infection. MHC class I-restricted presentation of these Ags suggests that these proteins or domains from them have access to the host cell cytoplasm. Chlamydia products with access to the host cell cytoplasm define a subset of molecules uniquely positioned to interface with the intracellular environment during the pathogen’s developmental cycle. In addition to their use as candidate Ags for stimulating CD8+ T cells, these proteins represent novel candidates for therapeutic intervention of infection. In this study, we use C. trachomatis-specific murine T cells and an expression-cloning strategy to show that CT442 from Chlamydia is targeted by CD8+ T cells. CT442, also known as CrpA, is a 15-kDa protein of undefined function that has previously been shown to be associated with the Chlamydia inclusion membrane. We show that: 1) CD8+ T cells specific for an H-2Db-restricted epitope from CrpA are elicited at a significant level (∼4% of splenic CD8+ T cells) in mice in response to infection; 2) the response to this epitope correlates with clearance of the organism from infected mice; and 3) immunization with recombinant vaccinia virus expressing CrpA elicits partial protective immunity to subsequent i.v. challenge with C. trachomatis.

The development of an effective vaccine against Mycobacterium tuberculosis is a research area of intense interest. Mounting evidence suggests that protective immunity to M. tuberculosis relies on both MHC class II-restricted CD4+ T cells and MHC class I-restricted CD8+ T cells. By purifying polypeptides present in the culture filtrate of M. tuberculosis and evaluating these molecules for their ability to stimulate PBMC from purified protein derivative-positive healthy individuals, we previously identified a low-m.w. immunoreactive T cell Ag, Mtb 8.4, which elicited strong Th1 T cell responses in healthy purified protein derivative-positive human PBMC and in mice immunized with recombinant Mtb 8.4. Herein we report that Mtb 8.4-specific T cells can be detected in mice immunized with the current live attenuated vaccine, Mycobacterium bovis-bacillus Calmette-Guérin as well as in mice infected i.v. with M. tuberculosis. More importantly, immunization of mice with either plasmid DNA encoding Mtb 8.4 or Mtb 8.4 recombinant protein formulated with IFA elicited strong CD4+ T cell and CD8+ CTL responses and induced protection on challenge with virulent M. tuberculosis. Thus, these results suggest that Mtb 8.4 is a potential candidate for inclusion in a subunit vaccine against TB.

Previous studies in murine and human models have suggested an important role for HLA Ia-restricted CD8+ T cells in host defense to Mycobacterium tuberculosis (Mtb). Therefore, understanding the Ags presented via HLA-Ia will be important in understanding the host response to Mtb and in rational vaccine design. We have used monocyte-derived dendritic cells in a limiting dilution analysis to generate Mtb-specific CD8+ T cells. Two HLA-Ia-restricted CD8+ T cell clones derived by this method were selected for detailed analysis. One was HLA-B44 restricted, and the other was HLA-B14 restricted. Both were found to react with Mtb-infected, but not bacillus Calmette-Guérin-infected, targets. For both these clones, the Ag was identified as culture filtrate protein 10 (CFP10)/Mtb11, a 10.8-kDa protein not expressed by bacillus Calmette-Guérin. Both clones were inhibited by the anti-class I Ab and anti-HLA-B,C Abs. Using a panel of CFP10/Mtb11-derived 15-aa peptides overlapping by 11 aa, the region containing the epitopes for both clones has been defined. Minimal 10-aa epitopes were defined for both clones. CD8+ effector cells specific for these two epitopes are present at high frequency in the circulating pool. Moreover, the CD8+ T cell response to CFP10/Mtb11 can be largely accounted for by the two epitopes defined herein, suggesting that this is the immunodominant response for this purified protein derivative-positive donor. This study represents the first time CD8+ T cells generated against Mtb-infected APC have been used to elucidate an Mtb-specific CD8+ T cell Ag.

Infection of C57BL/6 mice with Mycobacterium tuberculosis results in the development of a progressive disease during the first 2 wk after challenge. Thereafter, the disease is controlled by the emergence of protective T cells. We have used this infection model in conjunction with direct T cell expression cloning to identify Ags involved with the early control of the disease. A protective M. tuberculosis-specific CD4 T cell line derived from mice at 3 wk postchallenge was used to directly screen an M. tuberculosis genomic expression library. This screen resulted in the identification of a genomic clone comprising two putative adjacent genes with predicted open reading frames of 10 and 41 kDa, MTB10 and MTB41, respectively (the products of Rv0916c and Rv0915c, respectively, in the TubercuList H37Rv database). MTB10 and MTB41 belong to the PE and PPE family of proteins recently identified to comprise 10% of the M. tuberculosis genome. Evaluation of the recombinant proteins revealed that MTB41, but not MTB10, is the Ag recognized by the cell line and by M. tuberculosis-sensitized human PBMC. Moreover, C57BL/6 mice immunized with MTB41 DNA developed both CD4- (predominantly Th1) and CD8-specific T cell responses to rMTB41 protein. More importantly, immunization of C57BL/6 mice with MTB41 DNA induced protection against infection with M. tuberculosis comparable to that induced by bacillus Calmette-Guérin. Thus, the use of a proven protective T cell line in conjunction with the T cell expression cloning approach resulted in the identification of a candidate Ag for a subunit vaccine against tuberculosis.

In order to identify antigens that may be used in the serodiagnosis of active tuberculosis (TB), we screened a Mycobacterium tuberculosis genomic expression library with a pool of sera from patients diagnosed with active pulmonary TB. The sera used lacked reactivity with a recombinant form of the M. tuberculosis38-kDa antigen (r38kDa), and the goal was to identify antigens that might complement r38kDa in a serodiagnostic assay. Utilizing this strategy, we identified a gene, previously designated lhp, which encodes a 100-amino-acid protein referred to as culture filtrate protein 10 (CFP-10). The lhp gene is located directly upstream of esat-6, within a region missing in M. bovis BCG. Immunoblot analysis demonstrated that CFP-10 is present in M. tuberculosis CFP, indicating that it is likely a secreted or shed antigen. Purified recombinant CFP-10 (rCFP-10) was shown to be capable of detecting specific antibody in a percentage of TB patients that lack reactivity with r38kDa, most notably in smear-negative cases, where sensitivity was increased from 21% for r38kDa alone to 40% with the inclusion of rCFP-10. In smear-positive patient sera, sensitivity was increased from 49% for r38kDa alone to 58% with the inclusion of rCFP-10. In addition, rCFP-10 was shown to be a potent T-cell antigen, eliciting proliferative responses and gamma interferon production from peripheral blood mononuclear cells in 70% of purified protein derivative-positive individuals without evident disease. The responses to this antigen argue for the inclusion of rCFP-10 in a polyvalent serodiagnostic test for detection of active TB infection. rCFP-10 could also contribute to the development of a recombinant T-cell diagnostic test capable of detecting exposure to M. tuberculosis.

We have used expression screening of a genomic Mycobacterium tuberculosis library with tuberculosis (TB) patient sera to identify novel genes that may be used diagnostically or in the development of a TB vaccine. Using this strategy, we have cloned a novel gene, termed mtb39a , that encodes a 39-kDa protein. Molecular characterization revealed that mtb39a is a member of a family of three highly related genes that are conserved among strains of M. tuberculosis and Mycobacterium bovis BCG but not in other mycobacterial species tested. Immunoblot analysis demonstrated the presence of Mtb39A in M. tuberculosis lysate but not in culture filtrate proteins (CFP), indicating that it is not a secreted antigen. This conclusion is strengthened by the observation that a human T-cell clone specific for purified recombinant Mtb39A protein recognized autologous dendritic cells infected with TB or pulsed with purified protein derivative (PPD) but did not respond to M. tuberculosis CFP. Purified recombinant Mtb39A elicited strong T-cell proliferative and gamma interferon responses in peripheral blood mononuclear cells from 9 of 12 PPD-positive individuals tested, and overlapping peptides were used to identify a minimum of 10 distinct T-cell epitopes. Additionally, mice immunized with mtb39a DNA have shown increased protection from M. tuberculosis challenge, as indicated by a reduction of bacterial load. The human T-cell responses and initial animal studies provide support for further evaluation of this antigen as a possible component of a subunit vaccine for M. tuberculosis .