Thorsten Demberg

Multiple Vaccine-Elicited Nonneutralizing Antienvelope Antibody Activities Contribute to Protective Efficacy by Reducing both Acute and Chronic Viremia following Simian/Human Immunodeficiency Virus SHIV89.6P Challenge in Rhesus Macaques

ABSTRACT We have shown that following priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, boosting with gp140 envelope protein enhances acute-phase protection against intravenous simian/human immunodeficiency virus (SHIV)89.6P challenge compared to results with priming and no boosting or boosting with an HIV polypeptide representing the CD4 binding site of gp120. We retrospectively analyzed antibodies in sera and rectal secretions from these same macaques, investigating the hypothesis that vaccine-elicited nonneutralizing antibodies contributed to the better protection. Compared to other immunized groups or controls, the gp140-boosted group exhibited significantly greater antibody activities mediating antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated viral inhibition (ADCVI) in sera and transcytosis inhibition in rectal secretions. ADCC and ADCVI activities were directly correlated with antibody avidity, suggesting the importance of antibody maturation for functionality. Both ADCVI and percent ADCC killing prechallenge were significantly correlated with reduced acute viremia. The latter, as well as postchallenge ADCVI and ADCC, was also significantly correlated with reduced chronic viremia. We have previously demonstrated induction by the prime/boost regimen of mucosal antibodies that inhibit transcytosis of SIV across an intact epithelial cell layer. Here, antibody in rectal secretions was significantly correlated with transcytosis inhibition. Importantly, the transcytosis specific activity (percent inhibition/total secretory IgA and IgG) was strongly correlated with reduced chronic viremia, suggesting that mucosal antibody may help control cell-to-cell viral spread during the course of infection. Overall, the replicating Ad5hr-HIV/SIV priming/gp140 protein boosting approach elicited strong systemic and mucosal antibodies with multiple functional activities associated with control of both acute and chronic viremia.

Contribution of Nonneutralizing Vaccine-Elicited Antibody Activities to Improved Protective Efficacy in Rhesus Macaques Immunized with Tat/Env Compared with Multigenic Vaccines

Previously, chronic-phase protection against SHIV89.6P challenge was significantly greater in macaques primed with replicating adenovirus type 5 host range mutant (Ad5hr) recombinants encoding HIVtat and env and boosted with Tat and Env protein compared with macaques primed with multigenic adenovirus recombinants (HIVtat, HIVenv, SIVgag, SIVnef) and boosted with Tat, Env, and Nef proteins. The greater protection was correlated with Tat- and Env-binding Abs. Because the macaques lacked SHIV89.6P-neutralizing activity prechallenge, we investigated whether Ab-dependent cellular cytotoxicity (ADCC) and Ab-dependent cell-mediated viral inhibition (ADCVI) might exert a protective effect. We clearly show that Tat can serve as an ADCC target, although the Tat-specific activity elicited did not correlate with better protection. However, Env-specific ADCC activity was consistently higher in the Tat/Env group, with sustained cell killing postchallenge exhibited at higher levels (p < 0.00001) for a longer duration (p = 0.0002) compared with the multigenic group. ADCVI was similarly higher in the Tat/Env group and significantly correlated with reduced acute-phase viremia at wk 2 and 4 postchallenge (p = 0.046 and 0.011, respectively). Viral-specific IgG and IgA Abs in mucosal secretions were elicited but did not influence the outcome of the i.v. SHIV89.6P challenge. The higher ADCC and ADCVI activities seen in the Tat/Env group provide a plausible mechanism responsible for the greater chronic-phase protection. Because Tat is known to enhance cell-mediated immunity to coadministered Ags, further studies should explore its impact on Ab induction so that it may be optimally incorporated into HIV vaccine regimens.

A replication-competent adenovirus-human immunodeficiency virus (Ad-HIV) tat and Ad-HIV env priming/Tat and envelope protein boosting regimen elicits enhanced protective efficacy against simian/human immunodeficiency virus SHIV89.6P challenge in rhesus macaques.

ABSTRACT We previously demonstrated that replication-competent adenovirus (Ad)-simian immunodeficiency virus (SIV) recombinant prime/protein boost regimens elicit potent immunogenicity and strong, durable protection of rhesus macaques against SIVmac251. Additionally, native Tat vaccines have conferred strong protection against simian/human immunodeficiency virus SHIV89.6P challenge of cynomolgus monkeys, while native, inactivated, or vectored Tat vaccines have failed to elicit similar protective efficacy in rhesus macaques. Here we asked if priming rhesus macaques with replicating Ad-human immunodeficiency virus (HIV) tat and boosting with the Tat protein would elicit protection against SHIV89.6P. We also evaluated a Tat/Env regimen, adding an Ad-HIV env recombinant and envelope protein boost to test whether envelope antibodies would augment acute-phase protection. Further, expecting cellular immunity to enhance chronic viremia control, we tested a multigenic group: Ad-HIV tat, -HIV env, -SIV gag, and -SIV nef recombinants and Tat, Env, and Nef proteins. All regimens were immunogenic. A hierarchy was observed in enzyme-linked immunospot responses (with the strongest response for Env, followed by Gag, followed by Nef, followed by Tat) and antibody titers (with the highest titer for Env, followed by Tat, followed by Nef, followed by Gag). Following intravenous SHIV89.6P challenge, all macaques became infected. Compared to controls, no protection was seen in the Tat-only group, confirming previous reports for rhesus macaques. However, the multigenic group blunted acute viremia by approximately 1 log (P = 0.017), and both the multigenic and Tat/Env groups reduced chronic viremia by 3 and 4 logs, respectively, compared to controls (multigenic, P = 0.0003; Tat/Env, P < 0.0001). The strikingly greater reduction in the Tat/Env group than in the multigenic group (P = 0.014) was correlated with Tat and Env binding antibodies. Since prechallenge anti-Env antibodies lacked SHIV89.6P-neutralizing activity, other functional anti-Env and anti-Tat activities are under investigation, as is a possible synergy between the Tat and Env immunogens.

Mucosal immunity and protection against HIV/SIV infection: strategies and challenges for vaccine design.

To date, most HIV vaccine strategies have focused on parenteral immunization and systemic immunity. These approaches have not yielded the efficacious HIV vaccine urgently needed to control the AIDS pandemic. As HIV is primarily mucosally transmitted, efforts are being re-focused on mucosal vaccine strategies, in spite of complexities of immune response induction and evaluation. Here, we outline issues in mucosal vaccine design and illustrate strategies with examples from the recent literature. Development of a successful HIV vaccine will require in-depth understanding of the mucosal immune system, knowledge that ultimately will benefit vaccine design for all mucosally transmitted infectious agents.

Dynamics of Memory B-Cell Populations in Blood, Lymph Nodes, and Bone Marrow during Antiretroviral Therapy and Envelope Boosting in Simian Immunodeficiency Virus SIVmac251-Infected Rhesus Macaques

ABSTRACT Human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection causes B-cell dysregulation and the loss of memory B cells in peripheral blood mononuclear cells (PBMC). These effects are not completely reversed by antiretroviral treatment (ART). To further elucidate B-cell changes during chronic SIV infection and treatment, we investigated memory B-cell subpopulations and plasma cells/plasmablasts (PC/PB) in blood, bone marrow, and lymph nodes of rhesus macaques during ART and upon release from ART. Macaques previously immunized with SIV recombinants and the gp120 protein were included to assess the effects of prior vaccination. ART was administered for 11 weeks, with or without gp120 boosting at week 9. Naïve and resting, activated, and tissue-like memory B cells and PC/PB were evaluated by flow cytometry. Antibody-secreting cells (ASC) and serum antibody titers were assessed. No lasting changes in B-cell memory subpopulations occurred in bone marrow and lymph nodes, but significant decreases in numbers of activated memory B cells and increases in numbers of tissue-like memory B cells persisted in PBMC. Macaque PC/PB were found to be either CD27+ or CD27− and therefore were defined as CD19+ CD38hi CD138+. The numbers of these PC/PB were transiently increased in both PBMC and bone marrow following gp120 boosting of the unvaccinated and vaccinated macaque groups. Similarly, ASC numbers in PBMC and bone marrow of the two macaque groups also transiently increased following envelope boosting. Nevertheless, serum binding titers against SIVgp120 remained unchanged. Thus, even during chronic SIV infection, B cells respond to antigen, but long-term memory does not develop, perhaps due to germinal center destruction. Earlier and/or prolonged treatment to allow the generation of virus-specific long-term memory B cells should benefit ART/therapeutic vaccination regimens.

Vaccine-elicited SIV and HIV envelope-specific IgA and IgG memory B cells in rhesus macaque peripheral blood correlate with functional antibody responses and reduced viremia

An effective HIV vaccine requires strong systemic and mucosal, cellular and humoral immunity. Numerous non-human primate studies have investigated memory T cells, but not memory B cells. Humoral immunologic memory is mediated by long-lived antibody-secreting plasma cells and differentiation of memory B cells into short-lived plasma blasts following re-exposure to immunizing antigen. Here we studied memory B cells in vaccinated rhesus macaques. PBMC were stimulated polyclonally using CD40 Ligand, IL-21 and CpG to induce B cell proliferation and differentiation into antibody secreting cells (ASCs). Flow cytometry was used for phenotyping and evaluating proliferation by CFSE dilution. B cell responses were quantified by ELISPOT. Methodology was established using PBMC of vaccinated elite-controller macaques that exhibited strong, multi-functional antibody activities. Subsequently, memory B cells elicited by two replicating Ad-recombinant prime/envelope boost regimens were retrospectively evaluated pre- and post-SIV and SHIV challenges. The vaccine regimens induced SIV and HIV Env-specific IgG and IgA memory B cells. Prior to challenge, IgA memory B cells were more numerous than IgG memory B cells, reflecting the mucosal priming immunizations. Pre- and post-challenge memory B cells were correlated with functional antibody responses including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated viral inhibition (ADCVI) and transcytosis inhibition. Post-challenge, Env-specific IgG and IgA memory B cells were correlated with reduced chronic viremia. We conclude that functional antibody responses elicited by our prime/boost regimen were effectively incorporated into the memory B cell pool where they contributed to control of viremia following re-exposure to the immunizing antigen.

Comparative study of Tat vaccine regimens in Mauritian cynomolgus and Indian rhesus macaques : Influence of Mauritian MHC haplotypes on susceptibility/resistance to SHIV89.6p infection

Protection afforded by HIV Tat-based vaccines has differed in Indian rhesus and Mauritian cynomolgus macaques. We evaluated native Tat and Ad-HIVtat priming/Tat-boosting regimens in both species. Both vaccines were immunogenic. Only the Ad-tat regimen modestly reduced acute viremia in rhesus macaques after SHIV(89.6P) challenge. Confounding variables uncovered in Mauritian macaques included significant associations of susceptibility to infection with MHC class IB and class II H2 and H5 haplotypes, and resistance to infection with class IB haplotypes H3 and H6. Although protection here was limited, Tat-based vaccines incorporating other HIV components have shown greater efficacy. Combination strategies should be further explored.

Mucosal B Cells Are Associated with Delayed SIV Acquisition in Vaccinated Female but Not Male Rhesus Macaques Following SIVmac251 Rectal Challenge.

Many viral infections, including HIV, exhibit sex-based pathogenic differences. However, few studies have examined vaccine-related sex differences. We compared immunogenicity and protective efficacy of monomeric SIV gp120 with oligomeric SIV gp140 in a pre-clinical rhesus macaque study and explored a subsequent sex bias in vaccine outcome. Each immunization group (16 females, 8 males) was primed twice mucosally with replication-competent Ad-recombinants encoding SIVsmH4 env/rev, SIV239 gag and SIV239 nefΔ1–13 and boosted twice intramuscularly with SIVmac239 monomeric gp120 or oligomeric gp140 in MF59 adjuvant. Controls (7 females, 5 males) received empty Ad and MF59. Up to 9 weekly intrarectal challenges with low-dose SIVmac251 were administered until macaques became infected. We assessed vaccine-induced binding, neutralizing, and non-neutralizing antibodies, Env-specific memory B cells and plasmablasts/plasma cells (PB/PC) in bone marrow and rectal tissue, mucosal Env-specific antibodies, and Env-specific T-cells. Post-challenge, only one macaque (gp140-immunized) remained uninfected. However, SIV acquisition was significantly delayed in vaccinated females but not males, correlated with Env-specific IgA in rectal secretions, rectal Env-specific memory B cells, and PC in rectal tissue. These results extend previous correlations of mucosal antibodies and memory B cells with protective efficacy. The gp140 regimen was more immunogenic, stimulating elevated gp140 and cyclic V2 binding antibodies, ADCC and ADCP activities, bone marrow Env-specific PB/PC, and rectal gp140-specific IgG. However, immunization with gp120, the form of envelope immunogen used in RV144, the only vaccine trial to show some efficacy, provided more significant acquisition delay. Further over 40 weeks of follow-up, no gp120 immunized macaques met euthanasia criteria in contrast to 7 gp140-immunized and 2 control animals. Although males had higher binding antibodies than females, ADCC and ADCP activities were similar. The complex challenge outcomes may reflect differences in IgG subtypes, Fc glycosylation, Fc-R polymorphisms, and/or the microbiome, key areas for future studies. This first demonstration of a sex-difference in SIV vaccine-induced protection emphasizes the need for sex-balancing in vaccine trials. Our results highlight the importance of mucosal immunity and memory B cells at the SIV exposure site for protection.

Labeling Extracellular Vesicles for Nanoscale Flow Cytometry

Extracellular vesicles (EVs), including exosomes and microvesicles, are 30–800 nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be bright enough for one EV to be detected without the generation of label-associated artifacts. To identify a strategy that robustly labels individual EVs, we used nanoFACS, a high-resolution flow cytometric method that utilizes light scattering and fluorescence parameters along with sample enumeration, to evaluate various labels. Specifically, we compared lipid-, protein-, and RNA-based staining methods and developed a robust EV staining strategy, with the amine-reactive fluorescent label, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester, and size exclusion chromatography to remove unconjugated label. By combining nanoFACS measurements of light scattering and fluorescence, we evaluated the sensitivity and specificity of EV labeling assays in a manner that has not been described for other EV detection methods. Efficient characterization of EVs by nanoFACS paves the way towards further study of EVs and their roles in health and disease.

Sequential Priming with Simian Immunodeficiency Virus (SIV) DNA Vaccines, with or without Encoded Cytokines, and a Replicating Adenovirus-SIV Recombinant Followed by Protein Boosting Does Not Control a Pathogenic SIVmac251 Mucosal Challenge

ABSTRACT Previously, combination DNA/nonreplicating adenovirus (Ad)- or poxvirus-vectored vaccines have strongly protected against SHIV89.6P, DNAs expressing cytokines have modulated immunity elicited by DNA vaccines, and replication-competent Ad-recombinant priming and protein boosting has strongly protected against simian immunodeficiency virus (SIV) challenge. Here we evaluated a vaccine strategy composed of these promising components. Seven rhesus macaques per group were primed twice with multigenic SIV plasmid DNA with or without interleukin-12 (IL-12) DNA or IL-15 DNA. After a multigenic replicating Ad-SIV immunization, all groups received two booster immunizations with SIV gp140 and SIV Nef protein. Four control macaques received control DNA plasmids, empty Ad vector, and adjuvant. All vaccine components were immunogenic, but the cytokine DNAs had little effect. Macaques that received IL-15-DNA exhibited higher peak anti-Nef titers, a more rapid anti-Nef anamnestic response postchallenge, and expanded CD8CM T cells 2 weeks postchallenge compared to the DNA-only group. Other immune responses were indistinguishable between groups. Overall, no protection against intrarectal challenge with SIVmac251 was observed, although immunized non-Mamu-A*01 macaques as a group exhibited a statistically significant 1-log decline in acute viremia compared to non-Mamu-A*01 controls. Possible factors contributing to the poor outcome include administration of cytokine DNAs to sites different from the Ad recombinants (intramuscular and intratracheal, respectively), too few DNA priming immunizations, a suboptimal DNA delivery method, failure to ensure delivery of SIV and cytokine plasmids to the same cell, and instability and short half-life of the IL-15 component. Future experiments should address these issues to determine if this combination approach is able to control a virulent SIV challenge.