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Intranasal M2SR (M2-deficient Single Replication influenza virus) vaccine induces robust immune responses in animal models and human subjects. A high-throughput multiplexed platform was used to analyze hemagglutinin-specific mucosal antibody responses in adults after a single dose of H3N2 M2SR. Nasal swab specimens were analyzed for total and hemagglutinin-specific IgA. Significant, dose-dependent increases in mucosal antibody responses to vaccine-matched and drifted H3N2 hemagglutinin were observed in M2SR vaccinated subjects regardless of baseline serum and mucosal immune status. These data suggest that M2SR induces broadly cross-reactive mucosal immune responses which may provide better protection against drifted and newly emerging influenza strains., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: A controlled human infection model for assessing tuberculosis (TB) immunity can accelerate new vaccine development., Methods: In this phase 1 dose escalation trial, 92 healthy adults received a single intradermal injection of 2 × 106 to 16 × 106 colony-forming units of Bacillus Calmette-Guérin (BCG). The primary endpoints were safety and BCG shedding as measured by quantitative polymerase chain reaction, colony-forming unit plating, and MGIT BACTEC culture., Results: Doses up to 8 × 106 were safe, and there was evidence for increased BCG shedding with dose escalation. The MGIT time-to-positivity assay was the most consistent and precise measure of shedding. Power analyses indicated that 10% differences in MGIT time to positivity (area under the curve) could be detected in small cohorts (n = 30). Potential biomarkers of mycobacterial immunity were identified that correlated with shedding. Transcriptomic analysis uncovered dose- and time-dependent effects of BCG challenge and identified a putative transcriptional TB protective signature. Furthermore, we identified immunologic and transcriptomal differences that could represent an immune component underlying the observed higher rate of TB disease incidence in males., Conclusions: The safety, reactogenicity, and immunogenicity profiles indicate that this BCG human challenge model is feasible for assessing in vivo TB immunity and could facilitate the vaccine development process., Clinical Trials Registration: NCT01868464 (ClinicalTrials.gov)., Competing Interests: Potential conflicts of interest. D. F. H. receives personal fees for scientific advisory board service for Moderna and Poolbeg Pharma; M. J. M. performs laboratory research and holds clinical trials contracts with Lilly, Pfizer, and Sanofi and receives personal fees for scientific advisory board service from Merck, Meissa Vaccines, Inc, and Pfizer; N. G. R. receives funding from Merck, Sanofi Pasteur, Pfizer, Lilly, and Quidel to perform clinical research and serves as a safety consultant for ICON and Emmes LCC. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mycobacterium tuberculosis (Mtb)-specific γ9δ2 T cells secrete granzyme A (GzmA) protective against intracellular Mtb growth. However, GzmA-enzymatic activity is unnecessary for pathogen inhibition, and the mechanisms of GzmA-mediated protection remain unknown. We show that GzmA homodimerization is essential for opsonization of mycobacteria, altered uptake into human monocytes, and subsequent pathogen clearance within the phagolysosome. Although monomeric and homodimeric GzmA bind mycobacteria, only homodimers also bind cluster of differentiation 14 (CD14) and Toll-like receptor 4 (TLR4). Without access to surface-expressed CD14 and TLR4, GzmA fails to inhibit intracellular Mtb. Upregulation of Rab11FIP1 was associated with inhibitory activity. Furthermore, GzmA colocalized with and was regulated by protein disulfide isomerase AI (PDIA1), which cleaves GzmA homodimers into monomers and prevents Mtb inhibitory activity. These studies identify a previously unrecognized role for homodimeric GzmA structure in opsonization, phagocytosis, and elimination of Mtb in human monocytes, and they highlight PDIA1 as a potential host-directed therapy for prevention and treatment of tuberculosis, a major human disease., Competing Interests: Potential conflicts of interest. All authors declare that they have no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Influenza A (H7N9) has caused multiple disease waves with evidence of strain diversification. Optimal influenza A (H7N9) prime-boost vaccine strategies are unknown., Methods: We recruited participants who had received monovalent inactivated A/Shanghai/2/2013 (H7N9) vaccine (MIV) approximately 5 years earlier, as follows: MIV with MF59 (MF59 × 2 group), MIV with AS03 (AS03 × 2 group), unadjuvanted MIV (No Adj group), MIV with MF59 or AS03 followed by unadjuvanted MIV (Adjx1 group), and A/H7-naive (unprimed group). Participants were randomized to receive 1 dose of AS03-adjuvanted or unadjuvanted A/Hong Kong/125/2017 (H7N9) MIV and were followed for safety and immunogenicity using hemagglutination inhibition (HAI) and neutralizing antibody assays., Results: We enrolled 304 participants: 153 received the adjuvanted boost and 151 received the unadjuvanted boost. At 21 days postvaccination, the proportion of participants with HAI antibody titers against the boosting vaccine strain of ≥40 in the adjuvanted and unadjuvanted arms, respectively, were 88% and 49% in MF59 × 2 group, 89% and 75% in AS03 × 2 group, 59% and 20% in No Adj group, 94% and 55% in Adjx1group, and 9% and 11% in unprimed group., Conclusions: Serologic responses to a heterologous A(H7N9) MIV boost were highest in participants primed and boosted with adjuvant-containing regimens., Clinical Trials Registration: NCT03738241., Competing Interests: Potential conflicts of interest . I. Y.’s institution received funding to conduct clinical research unrelated to this manuscript from Moderna and Pfizer; and I. Y. consults for Merck and Sanofi-Pasteur. P. W. has received research funding unrelated to this manuscript from Pfizer and Sanofi; and has consulted for Pfizer. C. B. C. serves as a consultant to Pfizer, Moderna, GSK, and Vir. K. M. E. serves as consultant to Bionet and IBM; and member of the Data Safety and Monitoring Boards for Sanofi, X-4 Pharma, Seqirus, Moderna, Pfizer, Merck, and Roche. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: We evaluated the associations between baseline influenza virus-specific hemagglutination inhibition (HAI) and microneutralization (MN) titers and subsequent symptomatic influenza virus infection in a controlled human infection study., Methods: We inoculated unvaccinated healthy adults aged 18-49 years with an influenza A/California/04/2009/H1N1pdm-like virus (NCT04044352). We collected serial safety labs, serum for HAI and MN, and nasopharyngeal swabs for reverse-transcription polymerase chain reaction (RT-PCR) testing. Analyses used the putative seroprotective titer of ≥40 for HAI and MN. The primary clinical outcome was mild-to-moderate influenza disease (MMID), defined as ≥1 postchallenge positive qualitative RT-PCR test with a qualifying symptom/clinical finding., Results: Of 76 participants given influenza virus challenge, 54 (71.1%) experienced MMID. Clinical illness was generally very mild. MMID attack rates among participants with baseline titers ≥40 by HAI and MN were 64.9% and 67.9%, respectively, while MMID attack rates among participants with baseline titers <40 by HAI and MN were 76.9% and 78.3%, respectively. The estimated odds of developing MMID decreased by 19% (odds ratio, 0.81 [95% confidence interval, .62-1.06]; P = .126) for every 2-fold increase in baseline HAI. There were no significant adverse events., Conclusions: We achieved a 71.1% attack rate of MMID. High baseline HAI and MN were associated with protection from illness. Clinical Trials Registration. NCT04044352., Competing Interests: Potential conflicts of interest. J. R. O. reports grants to his institution from the National Science Foundation, Bill & Melinda Gates Foundation, Pfizer, NIH, and World Health Organization; consulting fees from Putnam; and participation on advisory boards for Pfizer, Seqirus, and Moderna, all outside the submitted work. D. I. B. reports consulting fees from Rational Vaccines and participation on an advisory board with Moderna and Dynavax, outside the submitted work. C. W. W. reports grants to his institution from Janssen and AstraZeneca, outside the submitted work. M. T. M. reports grants to his institution from NIH, outside the submitted work. R. W. F. reports grants to his institution from Pfizer, outside the submitted work, and clinical trial support from Pfizer. E. W. reports grants to his institution from Clinetic, Moderna, Pfizer, Seqirus, and Iliad Biotechnologies; consulting fees from Iliad Biotechnologies; and participation on the Vaxcyte advisory board. W. A. K. reports grants to her institution from Leidos, outside the submitted work; clinical trial support from Pfizer; subcontract from Leidos; and advisory board participation with the NIH. N. R. reports grants to her institution from Merck, Sanofi Pasteur, Quidel, Pfizer, and Lilly, outside the submitted work. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2023.)

Background: Influenza A(H5N1) virus and other avian influenza virus strains represent major pandemic threats. Like all influenza A virus strains, A(H5N1) viruses evolve rapidly. Innovative immunization strategies are needed to induce cross-protective immunity., Methods: Subjects primed with clade 1 H5 antigen, with or without adjuvant, and H5-naive individuals were boosted with clade 2 H5 antigen. The impact of priming on T cells capable of both proliferation and cytokine production after antigen restimulation was assessed., Results: Subjects previously vaccinated with clade 1 H5 antigen developed significantly enhanced clade 2 H5 cross-reactive T cell responses detectable 6 months after vaccination with clade 2 H5 antigen. Priming dose (15 µg vs 45 or 90 µg) had no effect on magnitude of heterotypic H5 T cell responses. In contrast, age at priming negatively modulated both the magnitude and duration of heterotypic H5 T cell responses. Elderly subjects developed significantly less heterotypic H5 T cell boosting, predominantly for T cells capable of cytokine production. Adjuvant had a positive albeit weaker effect than age. The magnitude of CD4(+) interferon-γ producing T cells correlated with H5 antibody responses., Conclusions: H5 heterotypic priming prior to onset of an A(H5N1) pandemic may increase magnitude and duration of immunity against a newly drifted pandemic H5 virus., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Background: Two doses of either trivalent live attenuated or inactivated influenza vaccines (LAIV and TIV, respectively) are approved for young children (≥ 24 months old for LAIV and ≥ 6 months old for TIV) and induce protective antibody responses. However, whether combinations of LAIV and TIV are safe and equally immunogenic is unknown. Furthermore, LAIV is more protective than TIV in children for unclear reasons., Methods: Children 6-35 months old were administered, 1 month apart, 2 doses of either TIV or LAIV, or combinations of LAIV and TIV in both prime/boost sequences. Influenza-specific antibodies were measured by hemagglutination inhibition (HAI), and T cells were studied in flow cytometric and functional assays. Highly conserved M1, M2, and NP peptides predicted to be presented by common HLA class I and II were used to stimulate interferon-γ enzyme-linked immunospot responses., Results: All LAIV and/or TIV combinations were well tolerated and induced similar HAI responses. In contrast, only regimens containing LAIV induced influenza-specific CD4(+), CD8(+), and γδ T cells, including T cells specific for highly conserved influenza peptides., Conclusions: Prime/boost combinations of LAIV and TIV in young children were safe and induced similar protective antibodies. Only LAIV induced CD4(+), CD8(+), and γδ T cells relevant for broadly protective heterosubtypic immunity., Clinical Trials Registration: NCT00231907.

Background: One strategy for improving anti-tuberculosis (TB) vaccination involves the use of recombinant bacille Calmette-Guérin (rBCG) overexpressing protective TB antigens. rBCG30, which overexpresses the Mycobacterium tuberculosis secreted antigen Ag85b, was the first rBCG shown to induce significantly greater protection against TB in animals than parental BCG., Methods: We report here the first double-blind phase 1 trial of rBCG30 in 35 adults randomized to receive either rBCG30 or parental Tice BCG intradermally. Clinical reactogenicity was assessed, and state-of-the-art immunological assays were used to study Ag85b-specific immune responses induced by both vaccines., Results: Similar clinical reactogenicity occurred with both vaccines. rBCG30 induced significantly increased Ag85b-specific T cell lymphoproliferation, interferon (IFN)-gamma secretion, IFN-gamma enzyme-linked immunospot responses, and direct ex vivo intracellular IFN-gamma responses. Additional flow cytometry studies measuring carboxyfluorescein succinimidyl ester dilution and intracellular cytokine production demonstrated that rBCG30 significantly enhanced the population of Ag85b-specific CD4(+) and CD8(+) T cells capable of concurrent expansion and effector function. More importantly, rBCG30 significantly increased the number of Ag85b-specific T cells capable of inhibiting intracellular mycobacteria., Conclusions: These results provide proof of principal that rBCG can safely enhance human TB immunity and support further development of rBCG overexpressing Ag85b for TB vaccination.

We developed a carboxyfluorescein succinimidyl ester (CFSE)-based flow-cytometric assay that can detect different subsets of vaccinia-specific T cells capable of both antigen-specific expansion and protective effector functions. Proliferation and effector functions were detected by CFSE dilution and intracellular staining, respectively. Absolute numbers of CD4(+)/CFSE(lo)/interferon (IFN)- gamma (+), CD8(+)/CFSE(lo)/IFN- gamma (+), CD8(+)/CFSE(lo)/granzyme A(+), and CD8(+)/CFSE(lo)/CD107a(+) T cells present after in vitro stimulation with live vaccinia were significantly higher in immunized individuals (P<.05). These CD4(+) and CD8(+) T cell increases were >2 log higher than increases detectable by standard lymphoproliferation and cytotoxicity assays. Vaccinia-specific CD8(+)/CFSE(lo)/IFN- gamma (+) and granzyme A(+) T cell responses were significantly correlated with the results of standard (51)Cr-release cytolytic assays (P<.05). Furthermore, vaccinia induced antigen-specific memory gamma delta T cells. We demonstrate that vaccinia induces robust memory effector CD4(+), CD8(+), and gamma delta T cells, all of which are relevant for protection against smallpox. CFSE-based flow-cytometric assays will be useful in evaluating cell-mediated immune responses induced by new smallpox vaccines.

The ability of 17 recombinant mycobacterial proteins, native antigen 85 complex, lipoarabinomannan (LAM), and Mycobacterium tuberculosis lysate to detect antibody responses induced by bacille Calmette-Guérin (BCG) vaccination and active tuberculosis infection were studied in enzyme-linked immunosorbent assays. Only LAM-reactive serum immunoglobulin G responses were significantly increased in both BCG-vaccinated patients and patients with active tuberculosis (P<.05), and oral BCG vaccination also induced significant increases in LAM-reactive secretory immunoglobulin A (P<.05). LAM-reactive antibody assays can serve as markers of humoral and mucosal immunity in future trials of BCG and newer attenuated mycobacterial vaccines.

Tuberculosis (TB) vaccine development is hindered by the lack of clear surrogate markers of protective human immunity to Mycobacterium tuberculosis. This study evaluated the hypothesis that immune-mediated inhibition of mycobacterial growth would more directly correlate with protective TB immunity than other immunologic responses. Bacille Calmette-Guérin (BCG) vaccination, known to induce partial protection against TB, was used as a model system to investigate mechanistic relationships among different parameters of antigen-specific immunity. Effects of primary and booster intradermal BCG vaccinations were assessed in 3 distinct assays of mycobacterial inhibition. Correlations between vaccine-induced growth inhibition and other immune responses were analyzed. BCG significantly enhanced all antigen-specific responses. Peak responses occurred at 2 months after boosting. Statistical analyses suggested that each assay measured unique aspects of mycobacterial immunity. Despite previous evidence that type 1 immune responses are essential for TB immunity, interferon-gamma production did not correlate with mycobacterial inhibition. These results have important implications for TB vaccine development.

Induction of human gammadelta T cells was investigated in subjects who were vaccinated with live recombinant canarypox virus expressing human immunodeficiency virus (HIV) proteins or soluble MN rgp120. Both canarypox and rgp120 induced antigen-specific lymphoproliferative and interferon (IFN)-gamma responses. However, only canarypox vaccination induced increased gammadelta T cell responses detectable after secondary in vitro expansion (P<.02). These enhanced gammadelta T cell responses were specific for canarypox but not HIV antigens. Canarypox-specific gammadelta T cells were predominantly Vgamma9(+) and produced intracellular and secreted IFN-gamma. gammadelta T cell lines generated from canarypox vaccinees responded to canarypox antigens but not to mycobacterial antigens shown previously to induce bacille Calmette-Guérin-specific gammadelta T cells. Furthermore, canarypox vaccinations were associated with significantly higher NK cell expansions (P=.02). Increased IFN-gamma production by gammadelta T and NK cells could enhance the induction of protective type 1 memory immunity. Thus, stimulation of gammadelta T cells might be an important feature of live vaccines.

We conducted a placebo-controlled, double-dose-escalation trial of oral bacille Calmette-Guérin (BCG) vaccination in 48 healthy volunteers. Seven of 32 BCG recipients became purified protein derivative (PPD)-positive after dose 1, and only 1 remained positive after dose 2, which suggests that oral BCG has inhibitory effects on delayed-type hypersensitivity (DTH) responses. Ten of the original placebo recipients and 11 oral BCG recipients were recruited to return for an intradermal BCG booster vaccination. Five of 10 original placebo recipients developed PPD responses >/=10 mm, but none of 11 oral BCG recipients developed PPD induration after they received an intradermal BCG booster (P<.05; Fisher's exact test). These results document persistent inhibitory effects of oral BCG vaccination on mycobacteria-specific DTH responses. Despite inhibition of DTH, oral BCG induced significant increases in mycobacteria-specific interferon (IFN)-gamma responses in peripheral blood mononuclear cells. More detailed studies of cytokine and homing molecule expression indicated that differential mucosal versus cutaneous trafficking may explain the dissociation between IFN-gamma and DTH responses.

We investigated the ability of T cells expanded with mycobacterial antigens from healthy purified protein derivative-reactive donors and bacille Calmette-Guérin (BCG)-vaccinated volunteers to inhibit intracellular growth of BCG. Peripheral blood mononuclear cells were incubated for 7 days with mycobacterial whole lysate, live BCG, tetanus toxoid as control antigen, or medium alone. Autologous monocytes were separated by plastic adherence, allowed to mature for 6 days, and infected with BCG before serving as target cells. Expanded effector cells were cocultured with target cells for 72 h. Cocultures were then treated with 0.2% saponin to lyse infected monocytes and release intracellular BCG. Quantities of viable BCG present in these lysates were studied by colony-forming unit counting and radiometric labeling. We reproducibly found that lymphocytes expanded with mycobacterial whole lysate or live BCG significantly inhibited the intracellular growth of BCG, compared with lymphocytes expanded with tetanus toxoid or rested in medium. In addition, BCG vaccination enhanced the ability of T cells to inhibit intracellular mycobacterial growth in 3 of 5 volunteers. This assay may be useful for estimates of protective immunity induced by tuberculosis vaccines in human trials.

Clinical, microbiological, and immunologic responses were evaluated in volunteers vaccinated intradermally with bacille Calmette-Guérin (BCG). Most volunteers (98%) developed ulcerative lesions that drained for a mean +/- SE of 4.3 +/- 0.29 weeks. Mycobacterial DNA was detected by a polymerase chain reaction-based amplification technique in biopsy specimens from BCG ulcers 2 weeks after vaccination and in blood specimens 3 days after vaccination. Mycobacteria were cultured from ulcer drainage 2 months after vaccination, demonstrating a prolonged potential risk of contact spread of the vaccine strain. The duration of ulcer drainage was inversely correlated with prevaccination lymphoproliferative (r = -0.515; P < .002) and interferon gamma (r = -0.841; P < .002) responses specific to mycobacteria and directly correlated with postvaccination increases in lymphoproliferative (r = 0.498; P < .002) and interferon gamma (r = 0.688; P < .02) responses specific to mycobacteria. These results demonstrate the clinical reactogenicity of BCG and the potential risk of contact spread of the vaccine strain and suggest that clinical reactogenicity is a trade-off for the induction of protective mycobacterial immunity.

Healthy volunteers were randomized to receive percutaneous or intradermal bacille Calmette-Guérin (BCG) vaccination. Delayed-type hypersensitivity (DTH) to tuberculin, as well as proliferative and interferon-gamma responses in peripheral blood mononuclear cells stimulated by whole cell lysates and culture filtrates of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare, were compared before and after vaccination. Positive DTH reactions were detected in 83% of intradermal and 40% of percutaneous BCG recipients 3 months after vaccination (P < .004). M. tuberculosis-specific proliferation was increased after intradermal BCG (P < .01) but not after percutaneous BCG compared with prevaccination responses. In addition, M. tuberculosis-specific interferon-gamma production was increased after intradermal BCG compared with both prevaccination responses (P < .04) and those measured after percutaneous BCG (P < .05). Predominant immune responses stimulated by BCG were directed against antigens present in mycobacterial whole cell lysate. These results indicate that the BCG vaccination route can affect both in vivo and in vitro immune responses.