Healthy elderly Singaporeans show no age-related humoral hyporesponsiveness nor diminished plasmablast generation in response to influenza vaccine.

ABSTRACT: Improving influenza vaccine efficacy is a priority to reduce the burden of influenza-associated morbidity and mortality. By careful selection of individuals based on health we show sustained response to influenza vaccination in older adults. Sustaining health in aging could be an important player in maintaining immune responses to influenza vaccination. TRIAL REGISTRATION: NCT03266237. Registered 30 August 2017, https://clinicaltrials.gov/ct2/show/NCT03266237.

Protection of outbred mice against a vaginal challenge by a Chlamydia trachomatis serovar E recombinant major outer membrane protein vaccine is dependent on phosphate substitution in the adjuvant.

Chlamydia trachomatis is the most common bacterial sexually-transmitted pathogen for which there is no vaccine. We previously demonstrated that the degree of phosphate substitution in an aluminum hydroxide adjuvant in a TLR-4-based C. trachomatis serovar E (Ser E) recombinant major outer membrane protein (rMOMP) formulation had an impact on the induced antibody titers and IFN-γ levels. Here, we have extended these observations using outbreed CD-1 mice immunized with C. trachomatis Ser E rMOMP formulations to evaluate the impact on bacterial challenge. The results confirmed that the rMOMP vaccine containing the adjuvant with the highest phosphate substitution induced the highest neutralizing antibody titers while the formulation with the lowest phosphate substitution induced the highest IFN-γ production. The most robust protection was observed in mice vaccinated with the formulation containing the adjuvant with the lowest phosphate substitution, as shown by the number of mice with positive vaginal cultures, number of positive cultures and number of C. trachomatis inclusion forming units recovered. This is the first report showing that vaccination of an outbred strain of mice with rMOMP induces protection against a vaginal challenge with C. trachomatis.

RNA-Seq Signatures Normalized by mRNA Abundance Allow Absolute Deconvolution of Human Immune Cell Types.

The molecular characterization of immune subsets is important for designing effective strategies to understand and treat diseases. We characterized 29 immune cell types within the peripheral blood mononuclear cell (PBMC) fraction of healthy donors using RNA-seq (RNA sequencing) and flow cytometry. Our dataset was used, first, to identify sets of genes that are specific, are co-expressed, and have housekeeping roles across the 29 cell types. Then, we examined differences in mRNA heterogeneity and mRNA abundance revealing cell type specificity. Last, we performed absolute deconvolution on a suitable set of immune cell types using transcriptomics signatures normalized by mRNA abundance. Absolute deconvolution is ready to use for PBMC transcriptomic data using our Shiny app (https://github.com/giannimonaco/ABIS). We benchmarked different deconvolution and normalization methods and validated the resources in independent cohorts. Our work has research, clinical, and diagnostic value by making it possible to effectively associate observations in bulk transcriptomics data to specific immune subsets.

Antibodies to PcpA and PhtD protect mice against Streptococcus pneumoniae by a macrophage- and complement-dependent mechanism.

Currently marketed Streptococcus pneumoniae (Spn) vaccines, which contain polysaccharide capsular antigens from the most common Spn serotypes, have substantially reduced pneumococcal disease rates but have limited coverage. A trivalent pneumococcal protein vaccine containing pneumococcal choline-binding protein A (PcpA), pneumococcal histidine triad protein D (PhtD), and detoxified pneumolysin is being developed to provide broader, cross-serotype protection. Antibodies against detoxified pneumolysin protect against bacterial pneumonia by neutralizing Spn-produced pneumolysin, but how anti-PhtD and anti-PcpA antibodies protect against Spn has not been established. Here, we used a murine passive protection sepsis model to investigate the mechanism of protection by anti-PhtD and anti-PcpA antibodies. Depleting complement using cobra venom factor eliminated protection by anti-PhtD and anti-PcpA monoclonal antibodies (mAbs). Consistent with a requirement for complement, complement C3 deposition on Spn in vitro was enhanced by anti-PhtD and anti-PcpA mAbs and by sera from PhtD- and PcpA-immunized rabbits and humans. Moreover, in the presence of complement, anti-PhtD and anti-PcpA mAbs increased uptake of Spn by human granulocytes. Depleting neutrophils using anti-Ly6G mAbs, splenectomy, or a combination of both did not affect passive protection against Spn, whereas depleting macrophages using clodronate liposomes eliminated protection. These results suggest anti-PhtD and anti-PcpA antibodies induced by pneumococcal protein vaccines protect against Spn by a complement- and macrophage-dependent opsonophagocytosis.

Influenza Vaccine-Induced Antibody Responses Are Not Impaired by Frailty in the Community-Dwelling Elderly With Natural Influenza Exposure.

Background: Elderly adults over 65 years of age are recommended to receive seasonal influenza vaccination as they are at a higher risk of infection and its complications than the younger community. The elderly are often stratified according to frailty status where frail individuals are more susceptible to adverse health outcomes than their non-frail counterparts, however, it is not known whether immunity induced by influenza vaccination is impaired in the frail elderly. Study Design: Two hundred and five elderly subjects of Chinese ethnicity in Singapore (mean age 73.3 ± 5.3 years, 128 females and 77 males) were administered the recommended trivalent inactivated 2013-14 seasonal influenza vaccine (Vaxigrip™) containing A/H1N1, A/H3N2, and B strains. The elderly subjects were stratified into three groups according to Fried's frailty criteria (59 frail, 85 pre-frail, 61 robust) and were also ranked by Rockwood's frailty index (RFI). Statistical associations were evaluated between frailty status and pre- and post-vaccination antibody titres in sera measured by Hemagglutination inhibition (HAI) and microneutralization (MN) assays. Immunological responses across frailty strata were also studied in terms of leukocyte cellular distribution, cytokine levels and gene expression. Results: Post-vaccination, 83.4% of the subjects seroconverted for A/H1N1, 80.5% for A/H3N2, and 81% for the B strain. The seroconversion rates were comparable across frailty groups (A/H1N1, ANOVA, p = 0.7910; A/H3N2, ANOVA, p = 0.8356, B, ANOVA, p = 0.9741). Geometric mean titres of HAI and MN as well as seroprotection rates were also similar in all three frailty groups and uncorrelated with RFI (Spearman, r = 0.023, p = 0.738). No statistically significant differences were observed between the frailty groups in vaccine-induced modulation of leukocyte populations, cytokine responses, and gene expression profiles of peripheral blood mononuclear cells (PBMCs). Whereas, post- and pre-vaccination HAI titres were positively correlated after adjusting for age and gender (A/H1N1, R2 = 0.216, p = 9.1e-11; A/H3N2, R2 = 0.166, p = 3.4e-8; B, R2 = 0.104, p = 3.1e-5). With most subjects lacking previous history of influenza vaccination, the pre-vaccination titres were likely due to natural exposure and seen to match the pattern of influenza subtype prevalence in the time period of vaccination. Conclusion: The majority of the elderly subjects seroconverted for seasonal influenza upon vaccination, and importantly, influenza vaccination-induced humoral immune responses and seroprotection were similar across the frailty strata, indicating that frail individuals may also benefit from influenza vaccination. Pre-existing antibodies due to natural exposure appeared to positively influence vaccine-induced antibody responses.

Phosphate substitution in an AlOOH - TLR4 adjuvant system (SPA08) modulates the immunogenicity of Serovar E MOMP from Chlamydia trachomatis.

Chlamydia trachomatis is one of the most common sexually transmitted pathogens and the development of an effective vaccine is highly desirable. The Major Outer Membrane Protein (MOMP) is one of the most abundant and immunogenic chlamydial proteins. Here we investigated the effects of phosphate substitution on the physicochemical and immunochemical properties of an experimental vaccine composed of serovar E recombinant MOMP (rMOMP) and a proprietary adjuvant system SPA08, consisting of aluminum oxyhydroxide (AlOOH) containing the TLR4 agonist E6020. An increase in phosphate substitution in the AlOOH component of the adjuvant markedly decreased the adsorptive coefficient and adsorptive capacity for both Ser E rMOMP and E6020. In vaccine formulations used for immunizations, phosphate substitution induced a decrease in the % adsorption of Ser E rMOMP without affecting the % adsorption of E6020. Immunogenicity studies in CD1 mice showed that an increase in phosphate substitution of the SPA08 adjuvant resulted in an increase in Ser E rMOMP-specific serum total IgG and IgG1 but not IgG2a titers. The degree of phosphate substitution in SPA08 also significantly increased in vitro neutralization concomitant with a decrease in proinflammatory cytokines secreted by Ser E rMOMP-restimulated splenocytes. Taken together, the results of these studies suggest that the degree of phosphate substitution in AlOOH greatly affects the adsorption of E6020 and Ser E rMOMP to AlOOH resulting in significant effects on vaccine-induced cellular and humoral responses.

A bivalent pneumococcal histidine triad protein D-choline-binding protein A vaccine elicits functional antibodies that passively protect mice from Streptococcus pneumoniae challenge.

Vaccines based on conserved pneumococcal proteins are being investigated because serotype coverage by pneumococcal polysaccharide and polysaccharide conjugate vaccines is incomplete and may eventually decrease due to serotype replacement. Here, we examined the functionality of human antibodies induced by a candidate bivalent choline-binding protein A- pneumococcal histidine triad protein D (PcpA-PhtD) vaccine. Pre- and post-immune sera from subjects who had been vaccinated with the PcpA-PhtD candidate vaccine were tested in an established passive protection model in which mice were challenged by intravenous injection with Streptococcus pneumoniae serotype 3 strain A66.1. Serum antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Bacterial surface binding by serum antibodies was determined by a flow cytometry-based assay. Sera from 20 subjects were selected based on low activity of pre-immune samples in the passive protection model. Bacterial surface binding correlated more strongly with anti-PcpA (0.87; p < 0.0001) than with anti-PhtD (0.71; p < 0.0001). The odds ratio for predicting survival in the passive protection assay was higher for the anti-PcpA concentration (470 [95% confidence interval (CI), 46.8 to >999.9]) than for the anti-PhtD concentration (3.4 [95% CI, 1.9 to 5.6]) or bacterial surface binding (9.4 [95% CI, 3.6 to 24.3]). Pooled post-immune serum also protected mice against a challenge with S. pneumoniae serotype 3 strain WU2. Both anti-PcpA and anti-PhtD antibodies induced by the bivalent candidate vaccine mediate protection against S. pneumoniae. The results also showed that the ELISA titer might be useful as a surrogate for estimating the functional activity of antibodies induced by pneumococcal protein vaccines.

Murine responses to recombinant MVA versus ALVAC vaccines against tumor-associated antigens, gp100 and 5T4.

Virally vectored cancer vaccines comprise a new form of immunotherapy that aim to generate anti-tumor immune responses with potential for tumor clearance and enhanced patient survival. Here, we compared 2 replication-deficient poxviruses modified vaccinia Ankara (MVA) and ALVAC(2) in their ability to induce antigen expression and immunogenicity of the tumor-associated antigens (TAAs) 5T4 and gp100. To facilitate the comparison, recombinant MVA-gp100M and ALVAC(2)-5T4 were constructed to complement existing ALVAC(2)-gp100M and MVA-5T4 vectors. Recombinant TAA expression in chicken embryo fibroblast cells was confirmed by Western blot analysis. 5T4 expression was approximately equal for both viruses, whereas ALVAC-derived gp100 was quickly degraded, at a time point when MVA-derived gp100 was still stable and expressed at high levels. Human leukocyte antigen-A2 transgenic mice were vaccinated with recombinant viruses and the CD8 T-cell responses elicited against each TAA were monitored by interferon-γ enzyme-linked immunospot. No 5T4 peptide responses were detected using splenocytes from mice vaccinated with either vector, whereas vaccination with MVA elicited a significantly higher gp100-specific response than ALVAC(2) at 10 PFU (P<0.001). In CD-1 mice, each vector elicited similar 5T4 antibody responses, whereas MVA was more potent and induced gp100 antibody responses at a lower immunization dose than ALVAC (P<0.001). In this study, immunogenicity varied depending on the viral vector used and reflected vector-associated differences in in vitro TAA expression and stability. These findings suggest that novel vector-transgene combinations must be assessed individually when designing vaccines, and that stability of vector-encoded proteins produced in vitro may be useful as a predictor for in vitro immunogenicity.

Preclinical qualification of a new multi-antigen candidate vaccine for metastatic melanoma.

New therapies are urgently required for the treatment of patients with melanoma. Here we describe the generation and preclinical evaluation of 3 new recombinant ALVAC(2) poxviruses vCP2264, vCP2291, and vCP2292 for their ability to induce the desired cellular immune responses against the encoded melanoma-associated antigens. This was done either in HLA-A2/K transgenic mice or using in vitro antigen-presentation studies. These studies demonstrated that the vaccine was able to induce HLA-A*0201-restricted T-cell responses against gp100 and NY-ESO-1, detectable directly ex vivo, in HLA-A2/K-transgenic mice. The in vitro antigen presentation studies, in the absence of appropriate animal models, demonstrated that target cells infected with the vaccine construct were lysed by MAGE-1, MAGE-3 or MART-1 peptide-specific T cells. These data indicate that ALVAC(2)-encoded melanoma-associated antigens can be properly processed and presented to induce antigen-specific cytotoxic T-cell responses. To enhance the immunogenicity of the melanoma antigens, a TRIad of COstimulatory Molecules (TRICOM) were also cloned into all 3 vectors. Increased in vitro proliferation and IFN-γ production was observed with all ALVAC(2) poxviruses encoding TRICOM, confirming the immune-enhancing effect of the ALVAC-encoded TRICOM. These studies demonstrated that all components of the vaccine were functionally active and provide a rationale for moving this candidate vaccine to the clinic.

Tolerance induction by intrathymic expression of P0.

Genetic deficiency or instability of myelin protein zero (P0) results in hereditary motor sensory neuropathy. In view of recent advances in gene therapy, substitution of the molecular defect may become realistic in the near future. Here we investigate the impact of genetic deficiency of P0 on selection of the autoreactive T cell repertoire in the corresponding mouse model. We show that P0 mRNA transcripts are expressed in thymic stroma, similar to other myelin proteins and that expression of intact P0 protein can be detected by Western blot. Using a library of overlapping 20mer peptides spanning the entire length of P0 and applying the ELISPOT technique, we detected a strong immune response toward P0 extracellular domain peptide aa 41-60 in P0(-/-) knockout mice, but not in heterozygous P0(+/-) or wild-type (wt) mice. In addition, one cryptic epitope and two subdominant epitopes of P0 were identified. Using P0(-/-) into wt bone marrow (BM) chimeras we found that P0 expression in the host suffices for full tolerance induction, which is in line with its presence in thymic stroma. However, repopulation of P0(-/-) mice with wt BM led to partial induction of tolerance, suggesting that BM derived cells can also express this protein. Our findings may have implications for secondary autoimmunity developing after gene therapy in hereditary neuropathies and other diseases with genetically determined protein deficiency, because the repaired protein will then represent a foreign, nontolerized Ag.