Cell Responses In Rhesus Macaques Research Articles

Multivalent antigen display on nanoparticle immunogens increases B cell clonotype diversity and neutralization breadth to pneumoviruses

Nanoparticles for multivalent display and delivery of vaccine antigens have emerged as a promising avenue for enhancing B cell responses to protein subunit vaccines. Here, we evaluated B cell responses in rhesus macaques immunized with prefusion-stabilized respiratory syncytial virus (RSV) F glycoprotein trimer compared with nanoparticles displaying 10 or 20 copies of the same antigen. We show that multivalent display skews antibody specificities and drives epitope-focusing of responding B cells. Antibody cloning and repertoire sequencing revealed that focusing was driven by the expansion of clonally distinct B cells through recruitment of diverse precursors. We identified two antibody lineages that developed either ultrapotent neutralization or pneumovirus cross-neutralization from precursor B cells with low initial affinity for the RSV-F immunogen. This suggests that increased avidity by multivalent display facilitates the activation and recruitment of these cells. Diversification of the B cell response by multivalent nanoparticle immunogens has broad implications for vaccine design.

Enhanced Cross-Reactive and Polyfunctional Effector-Memory T Cell Responses by ICVAX-a Human PD1-Based Bivalent HIV-1 Gag-p41 Mosaic DNA Vaccine.

ABSTRACTVaccine-induced protective T cell immunity is necessary for HIV-1 functional cure. We previously reported that rhesus PD1-Gag-based DNA vaccination sustained simian-human immunodeficiency virus (SHIV) suppression by inducing effector-memory CD8+ T cells. Here, we investigated a human PD1-Gag-based DNA vaccine, namely, ICVAX, for clinical translation. PD1-based dendritic cell targeting and mosaic antigenic designs were combined to generate the ICVAX by fusing the human soluble PD1 domain with a bivalent HIV-1 Gag-p41 mosaic antigen. The mosaic antigen was cross-reactive with patients infected with B, CRF07/08_BC, and CRF01_AE variants. In mice, ICVAX elicited stronger, broader, and more polyfunctional T cell responses than mosaic Gag-p41 alone, and suppressed EcoHIV infection more efficiently. In macaques, ICVAX elicited polyfunctional effector-memory T cell responses that targeted multiple nonoverlapping epitopes of the Gag-p41 antigen. Furthermore, ICVAX manufactured following good manufacturing practices proved potent immunogenicity in macaques after biannual homologous vaccination, warranting clinical evaluation of ICVAX as an immunotherapy against HIV-1.IMPORTANCE This study presents that ICVAX, a PD1-based DNA vaccine against HIV-1, could induce broad and polyfunctional T cell responses against different HIV-1 subtypes. ICVAX encodes a recombinant antigen consisting of the human soluble PD1 domain fused with two mosaic Gag-p41 antigens. The mosaic antigens cover more than 500 HIV-1 strains circulating in China including the subtypes B/B’, CRF01_AE, and CRF07/08_BC. In mice, ICVAX elicited stronger, broader, and more polyfunctional T cell responses, with better EcoHIV suppression than the nontargeting mosaic Gag-p41 DNA vaccine. Moreover, both lab-generated and GMP-grade ICVAX also elicited strong polyfunctional effector-memory T cell responses in rhesus macaques with good immunogenicity against multiple nonoverlapping epitopes of the Gag-p41 antigen. This study therefore highlights the great potential to translate the PD1-based DNA vaccine approach into clinical use, and opens up new avenues for alternative HIV-1 vaccine design for HIV-1 preventive and functional cure.

Vaccine nanodiscs plus polyICLC elicit robust CD8+ T cell responses in mice and non-human primates

Conventional cancer vaccines based on soluble vaccines and traditional adjuvants have produced suboptimal therapeutic efficacy in clinical trials. Thus, there is an urgent need for vaccine technologies that can generate potent T cell responses with strong anti-tumor efficacy. We have previously reported the development of synthetic high-density protein (sHDL) nanodiscs for efficient lymph node (LN)-targeted co-delivery of antigen peptides and CpG oligonucleotides (a Toll-like receptor-9 agonist). Here, we performed a comparative study in mice and non-human primates (NHPs) to identify an ideal vaccine platform for induction of CD8+ T cell responses. In particular, we compared the efficacy of CpG class B, CpG class C, and polyICLC (a synthetic double-stranded RNA analog, a TLR-3 agonist), each formulated with antigen-carrying sHDL nanodiscs. Here, we report that sHDL-Ag admixed with polyICLC elicited robust Ag-specific CD8+ T cell responses in mice, and when used in combination with α-PD-1 immune checkpoint inhibitor, sHDL-Ag + polyICLC eliminated large established (~100 mm3) MC-38 tumors in mice. Moreover, sHDL-Gag + polyICLC induced robust Simian immunodeficiency virus Gag-specific, polyfunctional CD8+ T cell responses in rhesus macaques and could further amplify the efficacy of recombinant adenovirus-based vaccine. Notably, while both sHDL-Ag-CpG-B and sHDL-Ag-CpG-C generated strong Ag-specific CD8+ T cell responses in mice, their results were mixed in NHPs. Overall, sHDL combined with polyICLC offers a strong platform to induce CD8+ T cells for vaccine applications.

OMIP-052: An 18-Color Panel for Measuring Th1, Th2, Th17, and Tfh Responses in Rhesus Macaques.

This 18-color panel was designed to measure vaccine-induced T cell responses in rhesus macaques by intracellular cytokine staining (ICS). It detects seven cytokines, IFN-γ, IL-2, IL-4, IL-5, IL-13, IL-17, and IL-21, two memory markers, CD45RA and CCR7, and four follicular helper T cell (Tfh) markers, CXCR3, CXCR5, ICOS, and PD-1 (Figure 1). While this panel was optimized for use on cryopreserved rhesus macaque peripheral blood mononuclear cells (PBMC) (Table 1), it can also be used on rhesus macaque tissue samples and human PBMC and tissue samples. Experimental vaccines are often tested in nonhuman primates (NHP) for immunogenicity and protection in preclinical trials before proceeding to clinical trials in humans. Historically, vaccine-induced T cell responses have been measured by IFN-γ ELISpot or intracellular cytokine staining (ICS) for IFN-γ, IL-2, and TNF. However, antibody levels, rather than Th1 responses, correlate with protection for many vaccines including the HIV vaccine used in the RV144 clinical trial 1. As such, measuring only Th1 responses may not be adequate for determining T cell correlates of protection or risk in vaccine studies. Therefore, we expanded our 11-color intracellular cytokine staining (ICS) panel, described in OMIP-005, to 18 colors in order to assess a fuller spectrum of T cell responses (Table 2). IL-17 To decrease the variability of the assay due to age-related differences in T cell differentiation, cytokine measurements can be reported as a frequency of memory T cells using the markers CD45RA and CCR7 2. Similarly, IL-21 can be reported as a frequency of Tfh using a combination of the markers CXCR3, CXCR5, ICOS, and PD-1. This combination of markers allows for the delineation of Tfh cells by all currently accepted methods 3-5. This 18-color rhesus macaque panel is an extension of the 11-color panel described in OMIP-005. The cytokines IL-4, IL-5, IL-13, IL-17, and IL-21 were added as well as the Tfh subset markers CXCR3, CXCR5, ICOS, and PD-1. To accommodate these additional markers, CD28 and TNF were removed. It is similar to the following human panels also used to measure antigen-specific responses by intracellular staining: OMIP-001, OMIP-008, OMIP-009, OMIP-14, OMIP-16, OMIP-22, OMIP-25. However, this panel is designed for staining rhesus macaque PBMC (using NHP cross-reactive antibody clones) and includes additional Tfh markers, as well as IL-5 and IL-13. It is also the only panel with this combination of seven cytokines. We thank Mario Roederer and Pratip K. Chattopadhyay for advice and help in developing and optimizing this panel. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Codelivery of Envelope Protein in Alum with MVA Vaccine Induces CXCR3-Biased CXCR5+ and CXCR5- CD4 T Cell Responses in Rhesus Macaques.

The goal of an HIV vaccine is to generate robust and durable protective Ab. Vital to this goal is the induction of CD4(+) T follicular helper (TFH) cells. However, very little is known about the TFH response to HIV vaccination and its relative contribution to magnitude and quality of vaccine-elicited Ab titers. In this study, we investigated these questions in the context of a DNA/modified vaccinia virus Ankara SIV vaccine with and without gp140 boost in aluminum hydroxide in rhesus macaques. In addition, we determined the frequency of vaccine-induced CD4(+) T cells coexpressing chemokine receptor, CXCR5 (facilitates migration to B cell follicles) in blood and whether these responses were representative of lymph node TFH responses. We show that booster modified vaccinia virus Ankara immunization induced a distinct and transient accumulation of proliferating CXCR5(+) and CXCR5(-) CD4 T cells in blood at day 7 postimmunization, and the frequency of the former but not the latter correlated with TFH and B cell responses in germinal centers of the lymph node. Interestingly, gp140 boost induced a skewing toward CXCR3 expression on germinal center TFH cells, which was strongly associated with longevity, avidity, and neutralization potential of vaccine-elicited Ab response. However, CXCR3(+) cells preferentially expressed the HIV coreceptor CCR5, and vaccine-induced CXCR3(+)CXCR5(+) cells showed a moderate positive association with peak viremia following SIV251 infection. Taken together, our findings demonstrate that vaccine regimens that elicit CXCR3-biased TFH cell responses favor Ab persistence and avidity but may predispose to higher acute viremia in the event of breakthrough infections.

Primate immune responses to HIV-1 Env formulated in the saponin-based adjuvant AbISCO-100 in the presence or absence of TLR9 co-stimulation.

Protein-based vaccines require adjuvants to achieve optimal responses. Toll-like receptor (TLR) 9 agonists were previously shown to improve responses to protein-based vaccines, such as the Hepatitis B virus vaccine formulated in alum. Here, we used CpG-C together with the clinically relevant saponin-based adjuvant AbISCO-100/Matrix-M (AbISCO), to assess if TLR9 co-stimulation would quantitatively or qualitatively modulate HIV-1 envelope glycoprotein (Env)-specific B and T cell responses in rhesus macaques. The macaques were inoculated with soluble Env trimers in AbISCO, with or without the addition of CpG-C, using an interval similar to the Hepatitis B virus vaccine. Following a comprehensive evaluation of antigen-specific responses in multiple immune compartments, we show that the Env-specific circulating IgG, memory B cells and plasma cells displayed similar kinetics and magnitude in the presence or absence of CpG-C and that there was no apparent difference between the two groups in the elicited HIV-1 neutralizing antibody titers or antigen-specific CD4+ T cell responses. Importantly, the control of SHIV viremia was significantly improved in animals from both Env-immunized groups relative to adjuvant alone controls, demonstrating the potential of AbISCO to act as a stand-alone adjuvant for Env-based vaccines.

Optimizing delivery of HIV-1 conserved region-derived immunogen for induction of T and B cell responses in rhesus macaques

Background The complexity of candidate HIV-1 vaccine formulations is increasing due to extreme challenges faced when trying to prevent or control HIV-1 infection. Methods Immunogen HIVconsv based on the most conserved regions of the HIV-1 proteome was used to explore combinations of seven distinct vaccines modalities in heterologous prime-boost regimens delivered to rhesus macaques to optimize induction of T cell and antibody responses. These include plasmid DNA (P), Semliky Forest virus replicons delivered as DNA (DREP; D) or virus particles (VREP; V), modified vaccinia virus Ankara (MVA; M), adenoviruses of human (HAdV-5; A) and chimpanzee origin (ChAdV-63; C) and adjuvanted synthetic long peptides (SLP; S). Results A number of observations were made. Thus, a very potent combination for induction of HIV-1-specific T cells was an adenovirus vector (A or C) followed by poxvirus M. S boost broadened T cell responses, but did not prime T cells efficiently. D was a stronger prime than P. PPP was the best prime for T cells, while PSS was best for induction of antibodies. Even very complex regimen PPPAMSSCMV continued to recruit new T cell clones into the response to a single epitope, although a ceiling for immunodominant responses was reached; subdominant responses could be boosted up to the last V delivery. Finally, PPSS, but not SSSS could protect 2/6 animals from SIVmac251 acquisition. Conclusion These results will guide initial design of human trials. So far, human studies in Oxford testing CM, PPPCM and PPPMC regimen concur with observations made in rhesus macaques.

Optimization and qualification of an 8-color intracellular cytokine staining assay for quantifying T cell responses in rhesus macaques for pre-clinical vaccine studies

Vaccination and SIV challenge of macaque species is the best animal model for evaluating candidate HIV vaccines in pre-clinical studies. As such, robust assays optimized for use in nonhuman primates are necessary for reliable ex vivo measurement of immune responses and identification of potential immune correlates of protection. We optimized and qualified an 8-color intracellular cytokine staining assay for the measurement of IFNγ, IL-2, and TNF from viable CD4 and CD8 T cells from cryopreserved rhesus macaque PBMC stimulated with peptides. After optimization, five laboratories tested assay performance using the same reagents and PBMC samples; similar results were obtained despite the use of flow cytometers with different configurations. The 8-color assay was then subjected to a pre-qualification study to quantify specificity and precision. These data were used to set positivity thresholds and to design the qualification protocol. Upon completion of the qualification study, the assay was shown to be highly reproducible with low inter-aliquot, inter-day, and inter-operator variability according to the qualification criteria with an overall variability of 20–40% for each outcome measurement. Thus, the 8-color ICS assay was formally qualified according to the ICH guidelines Q2 (R1) for specificity and precision indicating that it is considered a standardized/robust assay acceptable for use in pre-clinical trial immunogenicity testing.

P05-07. Evaluation of peripheral and bone marrow B cell responses in rhesus macaques after immunization with soluble HIV-1 gp140 trimers

Background Broadly neutralizing antibodies (bNAbs) against HIV-1 are not efficiently elicited by vaccination despite considerable efforts. bNAb elicitation is likely limited both by current envelope glycoproteins (Env) immunogens, which are not sufficient mimics of the native Env spike complex, and by the lack of immunization regimens that promote optimal development and maturation of B cell responses. So far, analyses from protein immunization studies focus mainly on serological antibody responses and less on responses at the B cell level. Here, we characterize frequencies of antigen-specific antibody secreting cells (ASC) and memory B cells in the periphery and bone marrow of rhesus macaques immunized with soluble gp140 trimers. These analyses provide a foundation for the design of improved immunization protocols to elicit bNAbs against HIV-1.

A single dose of HIV DNA vaccine induces long lasting potent HIV-specific T cell responses in Rhesus macaques

Background Use of HIV DNA vaccines in rodents revealed enthusiastic strong and long lasting immune responses (IR) specific to all expressed HIV antigens. However, ''le desarroi'' began when these constructs were used in human and nonhuman primates and their immunogenicity was found to be very weak. To face this desarroi, HIV DNA immunizations were rapidly associated with heterologous boosts with recombinant viruses and purified proteins. However, efforts in many labs were pursued to develop strategies that increase the immunogenicity of these vaccines. Among these strategies we have chosen to transpose as close as possible in Rhesus Macaques, the conditions of immunization we used in mice [1,2].

Induction of a virus-specific effector–memory CD4+ T cell response by attenuated SIV infection

We investigated simian immunodeficiency virus (SIV)-specific CD4+ T cell responses in rhesus macaques chronically infected with attenuated or pathogenic SIV strains. Analysis of SIVΔnef-infected animals revealed a relatively high frequency of SIV-specific CD4+ T cells representing 4–10% of all CD4+ T lymphocytes directed against multiple SIV proteins. Gag-specific CD4+ T cells in wild-type SIV-infected animals were 5–10-fold lower in frequency and inversely correlated with the level of plasma viremia. SIV-specific CD4+ cells from SIVΔnef animals were predominantly CD27−CD28−CD45RAlowCCR7−CCR5−, consistent with an effector–memory subset, and included a fully differentiated CD45RA+CCR7− subpopulation. In contrast, SIV-specific CD4+ T cells from SIV-infected animals were mostly CD27+CD28+CD45RA−CCR7+CCR5+, consistent with an early central memory phenotype. The CD45RA+CCR7−CD4+ subset from SIVΔnef animals was highly enriched for effector CD4+ T cells, as indicated by the perforin expression and up-regulation of the lysosomal membrane protein CD107a after SIV Gag stimulation. SIV-specific CD4+ T cells in attenuated SIV-infected animals were increased in frequency in bronchioalveolar lavage and decreased in lymph nodes, consistent with an effector–memory T cell population. The ability of SIVΔnef to induce a high frequency virus-specific CD4+ T cell response with direct effector function may play a key role in protective immunity produced by vaccination with attenuated SIV strains.

Modulation of Vaccine‐Induced Immune Responses to Hepatitis C Virus in Rhesus Macaques by Altering Priming before Adenovirus Boosting

Preventive and therapeutic vaccine strategies aimed at controlling hepatitis C virus (HCV) infection should mimic the immune responses observed in patients who control or clear HCV, specifically T helper (Th) type 1 and CD8+ cell responses to multiple antigens, including nonstructural protein (NS) 3. Given the experience with human immunodeficiency virus, the best candidates for this are based on DNA prime, pox, or adenovirus boost regimens. In rhesus macaques, we compared NS3-expressing DNA prime and adenovirus boost strategy with 2 alternative priming approaches aimed at modifying Th1 and CD8+ responses: DNA adjuvanted with interleukin (IL)-2- and -12-encoding plasmids or Semliki Forest virus (SFV). All prime-boost regimens elicited NS3-specific B and T cell responses in rhesus macaques, including CD8+ responses. SFV priming induced higher lymphoproliferation and longer Th1 memory responses. The use of IL-2- and IL-12-expressing vectors resulted in reduced Th2 and antibody responses, which led to increased Th1 skewing but not to an increase in the magnitude of the IFN- gamma and CD8+ responses. All strategies induced Th1 cellular responses to HCV NS3, with fine modulations depending on the different priming approaches. When they are developed for more HCV antigens, these strategies could be beneficial in therapeutic vaccine approaches.

Optimization of intracellular cytokine staining for the quantitation of antigen-specific CD4 + T cell responses in rhesus macaques

Standard proliferation assays used for analysis of CD4 + T cell function have significant shortcomings, including limited sensitivity, lack of truly quantitative readouts and significant variability. We have optimized an intracellular cytokine staining (ICS) assay in rhesus macaques which allows us to identify virus-specific CD4 + T cells at the single-cell level with high sensitivity while reducing background staining to a minimum. A variety of parameters were tested to determine the optimal experimental conditions necessary for the detection of antigen-specific CD4 + T cells in macaques. Central to our optimized protocol was the addition of cross-linked costimulatory anti-CD28 and anti-CD49d Mabs, a modification which resulted in up to threefold enhancement of the frequency of TNF-α-secreting CD4 + T cells following superantigen- or antigen-specific stimulation. The ICS protocol was also optimized with respect to antigen concentration and duration of antigenic stimulation. These modifications resulted in a convenient and highly reproducible assay with intra- and inter-assay variability of less than 10%. Although cryopreservation of PBMC generally led to a 40% to 80% decrease in the frequency of antigen-specific CD4 + T cells detected by ICS using stimulation with viral proteins, the use of overlapping peptide pools minimized the effects of cryopreservation on ICS responses. The use of more sensitive techniques such as ICS permits delineation of antigen-specific cells at the single cell level and should provide new insights into pathogen-specific immune responses in the rhesus macaque model.

Boosting of SIV-specific T cell responses in rhesus macaques that resist repeated intravaginal challenge with SIVmac251.

Despite repeated high-risk exposure to infectious HIV-1, some individuals remain HIV-1 seronegative and apparently uninfected. The use of nonhuman primate model systems to study SIVmac transmission may help to elucidate the factors responsible for protection in exposed, seronegative (ESN) humans. In an earlier vaccination study, three control rhesus macaques that were exposed to three sequential intravaginal challenges with pathogenic SIVmac251 failed to show evidence of infection after 5 years of observation. 51Cr release assay results suggested that these animals had low-level cytotoxic T lymphocyte responses to SIVmac proteins. We hypothesized that these responses might be an important component of protection from mucosal challenge. We performed an additional intravaginal challenge of all three macaques and monitored SIV-specific T cell responses in peripheral blood, using the sensitive enzyme-linked immunospot (ELISpot) assay. After the fourth challenge, one animal became infected; this animal did not mount a strong SIV-specific T cell response. Two other macaques remained uninfected as determined by peripheral blood mononuclear cell (PBMC) coculture, polymerase chain reaction (PCR), and branched DNA (bDNA) analysis of peripheral blood and lymphoid tissues, but demonstrated boosting of SIV-specific T cell responses after challenge. These results support a protective role for SIVmac-specific T cells in repeatedly exposed, persistently seronegative rhesus macaques.

A DNA/MVA-based candidate human immunodeficiency virus vaccine for Kenya induces multi-specific T cell responses in rhesus macaques

The minimum requirement for candidate human immunodeficiency virus (HIV) vaccines to enter clinical evaluation in humans should be their demonstrable immunogenicity in non-human primates: induction of antibodies neutralizing primary HIV isolates or elicitation of broad T cell-mediated immune responses. Here, we showed in rhesus macaques that the very same vaccines that had entered clinical trials in Oxford and Nairobi, plasmid pTHr.HIVA DNA and recombinant modified vaccinia virus Ankara MVA.HIVA in a prime-boost protocol (Hanke & McMichael, Nature Medicine 6, 951-955, 2000), induced cellular immune responses specific for multiple HIV-derived epitopes. This was demonstrated by using the intracellular cytokine staining and ELISPOT assays detecting interferon-gamma and pools of peptides employed in the clinical studies. These results have both boosted our expectations for the performance of these vaccines in humans and increased our confidence about the choice of these assays as the primary readouts in the on-going human trials.

ELISPOT assay for detection of peptide specific Interferon-γ secreting cells in rhesus macaques

A reliable procedure to measure antigen specific T cell responses in rhesus macaques is required to determine the efficacy of vaccines and immunotherapies. The currently available T cell assays are poorly quantifiable or technically difficult to perform. Classical 51Cr-release cytotoxic T cell (CTL) assays are cumbersome and difficult to quantitate reproducibly. Detection of specific T-cell using MHC-peptide tetrameric complexes is highly sensitive, but requires knowledge of MHC type and prior identification of T cell epitopes. We therefore developed a rhesus interferon-γ (IFN-γ) ELISPOT assay capable of detecting IFN-γ secretion in response to stimulation with pooled 20-mer peptides. Peripheral blood mononuclear cells (PBMCs) from rhesus monkeys immunized with a DNA vaccine and recombinant canary pox encoding the Plasmodium knowlesi circumsporozoite protein (PkCSP) were incubated with pools of peptides from PkCSP. Positive responses to peptide pools and individual peptides ranging from 100 to 450 spot forming cells (SFC)/10 6 PBMC were detected in four of four immunized monkeys and in zero of two control monkeys. In two monkeys studied in detail, the IFN-γ response was focussed on a single 20-mer peptide, QGDGANAGQPQAQGDGANAG, and was dependent on CD4 +, but not CD8 +, T cells. Background responses in control monkeys and preimmunization PBMCs ranged from 10 to 50 SFC/10 6 PBMC. The average within assay and between assay coefficients of variation (CV) for this peptide ELISPOT were 21.9 and 24.7%, respectively. This peptide IFN-γ assay will be a useful tool for evaluation of T cell responses in rhesus macaques.

New insights into evaluating effective T-cell responses to HIV.

Towards addressing questions related to HIV pathogenesis and vaccine design we are fortunate to have the availability of the SIV-infected rhesus macaque model. The strengths of this model which include a rapid rate of progression to AIDS and knowledge of the dose route and strain of the infecting virus complement studies in HIV-infected patients in which the reagents host genetics and access to samples are more extensive and better defined. Unfortunately there is currently still too little known about the antiviral immune responses in either system to directly and accurately compare their similarities and differences and to draw any definitive conclusions. Therefore the data and views presented herein will simply reflect what has recently been discovered in both humans and non-human primate studies. (excerpt)