Elicitation of Robust Tier 2 Neutralizing Antibody Responses in Nonhuman Primates by HIV Envelope Trimer Immunization Using Optimized Approaches.

The development of stabilized recombinant HIV envelope trimers that mimic the virion surface molecule has increased enthusiasm for a neutralizing antibody (nAb)-based HIV vaccine. However, there is limited experience with recombinant trimers as immunogens in nonhuman primates, which are typically used as a model for humans. Here, we tested multiple immunogens and immunization strategies head-to-head to determine their impact on the quantity, quality, and kinetics of autologous tier 2 nAb development. A bilateral, adjuvanted, subcutaneous immunization protocol induced reproducible tier 2 nAb responses after only two immunizations 8 weeks apart, and these were further enhanced by a third immunization with BG505 SOSIP trimer. We identified immunogens that minimized non-neutralizing V3 responses and demonstrated that continuous immunogen delivery could enhance nAb responses. nAb responses were strongly associated with germinal center reactions, as assessed by lymph node fine needle aspiration. This study provides a framework for preclinical and clinical vaccine studies targeting nAb elicitation.

Harnessing Activin A Adjuvanticity to Promote Antibody Responses to BG505 HIV Envelope Trimers.

T follicular helper (TFH) cells are powerful regulators of affinity matured long-lived plasma cells. Eliciting protective, long-lasting antibody responses to achieve persistent immunity is the goal of most successful vaccines. Thus, there is potential in manipulating TFH cell responses. Herein, we describe an HIV vaccine development approach exploiting the cytokine activin A to improve antibody responses against recombinant HIV Envelope (Env) trimers in non-human primates. Administration of activin A improved the magnitude of Env-specific antibodies over time and promoted a significant increase in Env-specific plasma cells in the bone marrow. The boost in antibody responses was associated with reduced frequencies of T follicular regulatory (TFR) cells and increased germinal center T follicular helper (GC-TFH) to TFR cell ratios. Overall, these findings suggest that adjuvants inducing activin A production could potentially be incorporated in future rational design vaccine strategies aimed at improving germinal centers, long-lived plasma cells, and sustained antibody responses.

Serum immunoglobulin G, M and A response to Cryptosporidium parvum in Cryptosporidium-HIV co-infected patients.

BACKGROUND: Cryptosporidium parvum, the protozoan parasite, causes a significant enteric disease in immunocompromised hosts such as HIV patients. The present study was aimed to compare serum IgG, IgM and IgA responses to crude soluble antigen of C. parvum in HIV seropositive and seronegative patients co-infected with Cryptosporidium and to correlate the responses with symptomatology. METHODS: Cryptosporidium parvum specific serum antibody (IgG, IgM and IgA) responses were assessed by ELISA in 11 HIV seropositive Cryptosporidium positive (Group I), 20 HIV seropositive Cryptosporidium negative (Group II), 10 HIV seronegative Cryptosporidium positive (Group III), 20 HIV seronegative Cryptosporidium negative healthy individuals (Group IV) and 25 patients with other parasitic diseases (Group V). RESULTS: A positive IgG and IgA antibody response was observed in significantly higher number of Cryptosporidium infected individuals (Gp I and III) compared to Cryptosporidium un-infected individuals (Gp II, IV and V) irrespective of HIV/immune status. Sensitivity of IgG ELISA in our study was found to be higher as compared to IgM and IgA ELISA. The number of patients with positive IgG, IgM and IgA response was not significantly different in HIV seropositive Cryptosporidium positive patients with diarrhoea when compared to patients without diarrhoea and in patients with CD4 counts <200 when compared to patients with CD4 counts >200 cells/microl. CONCLUSION: The study showed specific serum IgG and IgA production in patients infected with Cryptosporidium, both HIV seropositive and seronegative as compared to uninfected subjects suggesting induction of Cryptosporidium specific humoral immune response in infected subjects. However, there was no difference in number of patients with positive response in HIV seropositive or seronegative groups indicating that HIV status may not be playing significant role in modulation of Cryptosporidium specific antibody responses. The number of patients with positive IgG, IgM and IgA response was not significantly different in patients with or without history of diarrhoea thereby indicating that Cryptosporidium specific antibody responses may not be necessarily associated with protection from symptomatology.

Rapid Germinal Center and Antibody Responses in Non-human Primates after a Single Nanoparticle Vaccine Immunization.

The first immunization in a protein prime-boost vaccination is likely to be critical for how the immune response unfolds. Using fine needle aspirates (FNAs) of draining lymph nodes (LNs), we tracked the kinetics of the primary immune response in rhesus monkeys immunized intramuscularly (IM) or subcutaneously (s.c.) with an eOD-GT8 60-mer nanoparticle immunogen to facilitate clinical trial design. Significant numbers of germinal center B (BGC) cells and antigen-specific CD4 T cells were detectable in the draining LN as early as 7 days post-immunization and peaked near day 21. Strikingly, s.c. immunization results in 10-fold larger antigen-specific BGC cell responses compared to IM immunization. Lymphatic drainage studies revealed that s.c. immunization resulted in faster and more consistent axillary LN drainage than IM immunization. These data indicate robust antigen-specific germinal center responses can occur rapidly to a single immunization with a nanoparticle immunogen and vaccine drainage substantially impacts immune responses in local LNs.

Recurrent group A Streptococcus tonsillitis is an immunosusceptibility disease involving antibody deficiency and aberrant TFH cells.

"Strep throat" is highly prevalent among children, yet it is unknown why only some children develop recurrent tonsillitis (RT), a common indication for tonsillectomy. To gain insights into this classic childhood disease, we performed phenotypic, genotypic, and functional studies on pediatric group A Streptococcus (GAS) RT and non-RT tonsils from two independent cohorts. GAS RT tonsils had smaller germinal centers, with an underrepresentation of GAS-specific CD4+ germinal center T follicular helper (GC-TFH) cells. RT children exhibited reduced antibody responses to an important GAS virulence factor, streptococcal pyrogenic exotoxin A (SpeA). Risk and protective human leukocyte antigen (HLA) class II alleles for RT were identified. Lastly, SpeA induced granzyme B production in GC-TFH cells from RT tonsils with the capacity to kill B cells and the potential to hobble the germinal center response. These observations suggest that RT is a multifactorial disease and that contributors to RT susceptibility include HLA class II differences, aberrant SpeA-activated GC-TFH cells, and lower SpeA antibody titers.

Evaluation of staining techniques, antigen detection and nested PCR for the diagnosis of cryptosporidiosis in HIV seropositive and seronegative patients.

The study was designed to determine the efficacy of modified Ziehl-Neelsen (ZN), safranine methylene blue (SM) staining, antigen detection ELISA and a nested PCR assay (specific for Cryptosporidium parvum) for detection of Cryptosporidium in HIV seropositive and seronegative patients with diarrhoea. Cryptosporidium was detected in 10 (4.9%), 9 (4.4%), 39 (18.9%) and 27 (13.1%) of 206 HIV seropositive and 7 (4.6%), 6 (3.9%), 21 (13.7%) and 17 (11.1%) of 153 HIV seronegative patients by ZN staining, SM staining, antigen detection ELISA and PCR, respectively. None of the 50 apparently healthy control subjects was found to be infected with Cryptosporidium by any of the techniques. Based on the criteria of 'true positive' samples positive by at least any two techniques out of ZN staining, antigen detection and PCR, sensitivity of ZN and SM staining techniques was 37% and 33.3% in HIV seropositive and 41.2% and 35.3% in seronegative patients, respectively. Sensitivity of antigen detection ELISA was 92.6% and 94.1% in HIV seropositive and seronegative patients, respectively, while sensitivity of PCR was 100% each in HIV seropositive and seronegative patients. Specificity of all three techniques, i.e. ZN, SM staining and PCR was 100% in both HIV seropositive and seronegative patients while specificity of antigen detection was 92.2% and 96.3% in HIV seropositive and seronegative patients, respectively. The staining techniques were found less sensitive as compared to antigen detection and PCR for detection of Cryptosporidium in HIV seropositive patients with CD4 count >200cells/microl.

The human naive B cell repertoire contains distinct subclasses for a germline-targeting HIV-1 vaccine immunogen.

Traditional vaccine development to prevent some of the worst current pandemic diseases has been unsuccessful so far. Germline-targeting immunogens have potential to prime protective antibodies (Abs) via more targeted immune responses. Success of germline-targeting vaccines in humans will depend on the composition of the human naive B cell repertoire, including the frequencies and affinities of epitope-specific B cells. However, the human naive B cell repertoire remains largely undefined. Assessment of antigen-specific human naive B cells among hundreds of millions of B cells from multiple donors may be used as pre-phase 1 ex vivo human testing to potentially forecast B cell and Ab responses to new vaccine designs. VRC01 is an HIV broadly neutralizing Ab (bnAb) against the envelope CD4-binding site (CD4bs). We characterized naive human B cells recognizing eOD-GT8, a germline-targeting HIV-1 vaccine candidate immunogen designed to prime VRC01-class Abs. Several distinct subclasses of VRC01-class naive B cells were identified, sharing sequence characteristics with inferred precursors of known bnAbs VRC01, VRC23, PCIN63, and N6. Multiple naive B cell clones exactly matched mature VRC01-class bnAb L-CDR3 sequences. Non-VRC01-class B cells were also characterized, revealing recurrent public light chain sequences. Unexpectedly, we also identified naive B cells related to the IOMA-class CD4bs bnAb. These different subclasses within the human repertoire had strong initial affinities (KD) to the immunogen, up to 13 nM, and represent encouraging indications that multiple independent pathways may exist for vaccine-elicited VRC01-class bnAb development in most individuals. The frequencies of these distinct eOD-GT8 B cell specificities give insights into antigen-specific compositional features of the human naive B cell repertoire and provide actionable information for vaccine design and advancement.

Direct Probing of Germinal Center Responses Reveals Immunological Features and Bottlenecks for Neutralizing Antibody Responses to HIV Env Trimer.

Generating tier 2 HIV-neutralizing antibody (nAb) responses by immunization remains a challenging problem, and the immunological barriers to induction of such responses with Env immunogens remain unclear. Here, some rhesus monkeys developed autologous tier 2 nAbs upon HIV Env trimer immunization (SOSIP.v5.2) whereas others did not. This was not because HIV Env trimers were immunologically silent because all monkeys made similar ELISA-binding antibody responses; the key difference was nAb versus non-nAb responses. We explored the immunological barriers to HIV nAb responses by combining a suite of techniques, including longitudinal lymph node fine needle aspirates. Unexpectedly, nAb development best correlated with booster immunization GC B cell magnitude and Tfh characteristics of the Env-specific CD4 T cells. Notably, these factors distinguished between successful and unsuccessful antibody responses because GC B cell frequencies and stoichiometry to GC Tfh cells correlated with nAb development, but did not correlate with total Env Ab binding titers.

Anti-streptolysin O titers in normal healthy children of 5-15 years.

Antistreptolysin O (ASO) levels vary with age group of the study population and geographical locations. The present study was undertaken to determine the upper limit of normal of ASO in 200 normal children of 5-15 years of age with no history of recent sore throat infection. The standard tube dilution method (WHO) was used for estimating ASO titers. It was found that 239 IU was the upper limit of normal in the study population, which can be considered as the baseline ASO titer. This can provide useful guidelines for physicians in the interpretation of elevated ASO titers in cases of suspected acute rheumatic fever.

Lymphoproliferative and cytokine responses to Cryptosporidium parvum in patients coinfected with C. parvum and human immunodeficiency virus.

We compared the lymphoproliferative and cytokine responses to Cryptosporidium parvum in human immunodeficiency virus (HIV)-seropositive and -seronegative patients. The lymphoproliferative and cytokine responses (interleukin-2 [IL-2], IL-4, IL-5, IL-10, gamma interferon, and tumor necrosis factor alpha) were assessed for 11 HIV-seropositive, Cryptosporidium-positive (group I) patients; 20 HIV-seropositive, Cryptosporidium-negative (group II) patients; 10 HIV-seronegative, Cryptosporidium-positive (group III) patients, including four post-renal transplant (group IIIa) and 6 presumably immunocompetent (group IIIb) patients; and 20 HIV-seronegative, Cryptosporidium-negative healthy individuals (group IV). No significant difference was observed in the number of patients showing positive lymphoproliferative responses in group I compared to group III (post-renal transplant [group IIIa] or immunocompetent [group IIIb]) patients, while a comparison of the median stimulation indices shows that responses were significantly lower in Cryptosporidium-infected, immunosuppressed (group I and IIIa) patients than in immunocompetent (group IIIb) patients. The number of patients showing positive responses and median stimulation indices was significantly higher for Cryptosporidium-infected (HIV-seropositive and -seronegative) individuals than for uninfected individuals, suggesting that Cryptosporidium induces significant in vitro lymphoproliferative responses in infected individuals. Cytokine levels, except for that of IL-5, were significantly higher in Cryptosporidium-infected (groups I and III) individuals than in uninfected (groups II and IV) individuals. There was no significant difference between the group I and III patients and between Cryptosporidium-infected immunosuppressed (group I or IIIa) and immunocompetent (group IIIb) patients.