[Show abstract][Hide abstract] ABSTRACT: Influenza viruses are promising mucosal vaccine vectors for HIV but their use has been limited by difficulties in engineering the expression of large amounts of foreign protein. We developed recombinant influenza viruses incorporating the HIV-1 p24 gag capsid into the NS-segment of PR8 (H1N1) and X31 (H3N2) influenza viruses with the use of multiple 2A ribosomal skip sequences. Despite the insertion of a sizable HIV-1 gene into the influenza genome, recombinant viruses were readily rescued to high titers. Intracellular expression of p24 capsid was confirmed by in vitro infection assays. The recombinant influenza viruses were subsequently tested as mucosal vaccines in BALB/c mice. Recombinant viruses were attenuated and safe in immunized mice. Systemic and mucosal HIV-specific CD8 T-cell responses were elicited in mice that were immunized via intranasal route with a prime-boost regimen. Isolated HIV-specific CD8 T-cells displayed polyfunctional cytokine and degranulation profiles. Mice boosted via intravaginal route induced recall responses from the distal lung mucosa and developed heightened HIV-specific CD8 T-cell responses in the vaginal mucosa. These findings demonstrate the potential utility of recombinant influenza viruses as vaccines for mucosal immunity against HIV-1 infection.
[Show abstract][Hide abstract] ABSTRACT: We report the engineering of poly(ethylene glycol) (PEG) hydrogel particles using a mesoporous silica (MS) templating method via tuning the PEG molecular weight, particle size, and the presence or absence of the template and investigate the cell association and biodistribution of these particles. An ex vivo assay based on human whole blood that is more sensitive and relevant than traditional cell-line based assays for predicting in vivo circulation behavior is introduced. The association of MS@PEG particles (template present) with granulocytes and monocytes is higher compared with PEG particles (template absent). Increasing the PEG molecular weight (from 10 to 40 kDa) or decreasing the PEG particle size (from 1400 to 150 nm) reduces phagocytic blood cell association of the PEG particles. Mice biodistribution studies show that the PEG particles exhibit extended circulation times (>12 h) compared with the MS@PEG particles and that the retention of smaller PEG particles (150 nm) in blood, when compared with larger PEG particles (>400 nm), is increased at least 4-fold at 12 h after injection. Our findings highlight the influence of unique aspects of polymer hydrogel particles on biological interactions. The reported PEG hydrogel particles represent a new class of polymer carriers with potential biomedical applications.
[Show abstract][Hide abstract] ABSTRACT: Yearly vaccination with the trivalent inactivated influenza vaccine (TIV) is recommended since current vaccines induce little cross neutralization to divergent influenza strains. Whether the TIV can induce ADCC responses that can cross-recognize divergent influenza strains is unknown. We immunized 6 influenza-naïve pigtail macaques twice with the 2011-2012 season TIV and then challenged macaques, along with 12 control macaques, serially with H1N1 and H3N2 viruses. We measured ADCC responses in plasma to a panel of H1 and H3 hemagglutinin (HA) proteins and influenza-specific CD8 T cell (CTL) responses using a sensitive MHC-tetramer reagent. The TIV was weakly immunogenic and, although binding antibodies were detected by ELISA, did not induce detectable influenza-specific ADCC or CTLs responses. The H1N1 challenge elicited robust ADCC to both homologous and heterologous H1 HA proteins but not influenza HA proteins from different subtypes (H2-H7). There was no anamnestic influenza-specific ADCC or CTL response in vaccinated animals. The subsequent H3N2 challenge did not induce or boost ADCC either to H1 HA proteins or divergent H3 proteins but did boost CTL responses. ADCC or CTLs responses are not induced by TIV vaccination in influenza-naïve macaques. There was a marked difference in the ability of infection compared to vaccination in inducing cross-reactive ADCC and CTL responses. Improved vaccination strategies are needed to induce broad-based ADCC immunity to influenza.
Full-text · Article · Oct 2013 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: DNA-loaded polypeptide particles are prepared via templated assembly of mesoporous silica for the delivery of adjuvants. The elasticity and cargo-loading capacity of the obtained particles can be tuned by the amount of cross-linker used to stabilize the polypeptide particles. The use of polypeptide particles as biocarriers provides a promising method for vaccine delivery.
[Show abstract][Hide abstract] ABSTRACT: Lymphoid tissues are of intense interest for studies of the pathogenesis of human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques but are relatively difficult to sample non-invasively. Fine needle aspiration (FNA) cytology, conventionally a diagnostic procedure for lymphadenopathy, can be used for longitudinal study of tissue cell subsets during HIV/SIV infection. In this study, we serially sampled lymph node (LN) FNA from pigtail macaques and studied cell subsets in the aspect of absolute count, frequency, and functionality by flow cytometry. The median recovered lymphocyte count from FNA samples was 2.01×10(5) (3.0×10(3) to 2.25×10(6), n = 38) and median CD4+ T cell subset recovered was 5.94×10(4) (277 to 6.17×10(5), n = 38). Although we observed a relatively large variation in the frequencies of cell subsets of FNA samples taken from different time points, the cell subset composition of FNA samples, in particular T cell and CD4+ T cell frequencies, was broadly comparable to whole excised LNs (n = 6) and distinct from peripheral blood. A subset of CD4+ T cells that is located almost exclusively in secondary lymphoid tissues, T follicular helper (TFH) cells, were readily identifiable in LN FNAs and the TFH cell frequencies were strongly correlated with B cell frequencies. In vitro functionality of FNA lymphocytes was demonstrated using polyclonal SEB stimulation, resulting in a median 6% of responding CD4+ T cells, comparable to circulating CD4+ T lymphocytes. We conclude that serial sampling of macaque LNs using FNA is a potentially useful method to study the immunopathogenesis of SIV infection and may be extended to HIV infection.
No preview · Article · May 2013 · Journal of immunological methods
[Show abstract][Hide abstract] ABSTRACT: There is an urgent need for a human immunodeficiency virus (HIV) vaccine that induces robust mucosal immunity. CD8+ cytotoxic T lymphocytes (CTLs) apply substantial antiviral pressure, but CTLs to individual epitopes select for immune escape
variants in both HIV in humans and SIV in macaques. Inducing multiple simian immunodeficiency virus (SIV)-specific CTLs may
assist in controlling viremia. We vaccinated 10 Mane-A1*08401+ female pigtail macaques with recombinant influenza viruses expressing three Mane-A1*08401-restricted SIV-specific CTL epitopes and subsequently challenged the animals, along with five controls, intravaginally with
SIVmac251. Seroconversion to the influenza virus vector resulted and small, but detectable, SIV-specific CTL responses were induced.
There was a boost in CTL responses after challenge but no protection from high-level viremia or CD4 depletion was observed.
All three CTL epitopes underwent a coordinated pattern of immune escape during early SIV infection. CTL escape was more rapid
in the vaccinees than in the controls at the more dominant CTL epitopes. Although CTL escape can incur a “fitness” cost to
the virus, a putative compensatory mutation 20 amino acids upstream from an immunodominant Gag CTL epitope also evolved soon
after the primary CTL escape mutation. We conclude that vaccines based only on CTL epitopes will likely be undermined by rapid
evolution of both CTL escape and compensatory mutations. More potent and possibly broader immune responses may be required
to protect pigtail macaques from SIV.
Full-text · Article · Jan 2013 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: T follicular helper (Tfh) cells are a specialized subset of memory CD4+ T cells that are exclusively found within the germinal centres of secondary lymphoid tissues, and are important for adaptive antibody responses and B cell memory. Tfh cells do not express CCR5, the primary entry co-receptor for both HIV-1 and SIV, and therefore we hypothesized that these cells would avoid infection. We studied lymph nodes and spleens from pigtail macaques infected with pathogenic strains, SIVmac239 or SIVmac251, to investigate the susceptibility of Tfh cells to SIV infection. Pigtail macaque PD-1(high)CD127(low) memory CD4+ T cells have a phenotype comparable to that of human Tfh cells, expressing high levels of CXCR5, IL-21, Bcl-6 and ICOS. As judged by either proviral DNA or cell-associated viral RNA measurements, macaque Tfh cells were infected with SIV at levels comparable to other CD4+ memory T cells. Infection of macaque Tfh cells was evident within weeks of inoculation, yet we confirmed that Tfh cells do not express CCR5, nor either of the well-known alternative SIV co-receptors, CXCR6 and Gpr15. Mutations in SIV envelope gp120 region occurred in chronically infected macaques but were uniform across each T cell subset investigated, indicating that the viruses used the same co-receptors to enter different cell subsets. Early infection of Tfh cells represents an unexpected focus of viral infection. Infection of Tfh cells does not interrupt antibody production, but may be a factor that limits the quality of antibody responses and has implications for assessing the size of the viral reservoir.
Full-text · Article · Jan 2013 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Defining which cells become infected with simian immunodeficiency virus (SIV) in vivo should assist in unravelling the pathogenesis of human immunodeficiency virus (HIV)/SIV infection. HIV/SIV infection of CD4(+) T cells resulted in down-regulation of CD3 and CD4 surface molecules in vitro, however this phenomenon is poorly characterised in vivo. Intracellular SIV p27 was studied by flow cytometry in serial blood samples and lymph node samples during acute infection of 17 SIVmac-infected pigtail macaques. Two weeks after infection, a mean of 56±6.8% the p27(+) cells were lymphocytes negative for surface CD4 and CD3, and indeed the highest proportion of SIV infected cells were found in the small subset of CD3(Lo)CD4(-)CD8(-) lymphocytes, indicating that infection has lead to down-regulation of these markers in vivo. Furthermore, the relative amount of SIV p27 within lymphocytes (based of mean fluorescence intensity) was higher in CD3(Lo)CD4(-) and CD3(-) infected cells than in CD3(+) or CD4(+) p27(+) populations, consistent with greater viral production in CD4(+) T cells down-regulating CD3 and CD4 molecules. The CD3(-)CD4(-) infected cells expressed T cell markers CD2 and CD5 and were negative for monocyte, NK and B cell markers. The majority of infected cells were CD28(+)CD95(+) central memory T cells. Surprisingly, p27(+) blood lymphocytes were mostly negative for activation markers CD25 and CD69, but most of the infected lymph nodes cells were activated. Our results characterise productively-infected macaque lymphocytes in vivo. The high proportion of SIV-infected lymphocytes that are CD3(-)CD4(-) has important implications for the in vivo study of pathogenesis of SIV/HIV infections.
[Show abstract][Hide abstract] ABSTRACT: Successful vaccination against HIV should limit viral replication sufficiently to prevent the emergence of viral immune escape mutations. Broadly directed immunity is likely to be required to limit opportunities for immune escape variants to flourish. We studied the emergence of an SIV Gag cytotoxic T cell immune escape variant in pigtail macaques expressing the Mane-A*10 MHC I allele using a quantitative RT-PCR to measure viral loads of escape and wild type variants. Animals receiving whole Gag expressing vaccines completely controlled an SIV(mac251) challenge, had broader CTL responses and exhibited minimal CTL escape. In contrast, animals vaccinated with only a single CTL epitope and challenged with the same SIV(mac251) stock had high levels of viral replication and rapid CTL escape. Unvaccinated naïve animals exhibited a slower emergence of immune escape variants. Thus narrowly directed vaccination against a single epitope resulted in rapid immune escape and viral levels equivalent to that of naïve unvaccinated animals. These results emphasize the importance of inducing broadly directed HIV-specific immunity that effectively quashes early viral replication and limits the generation of immune escape variants. This has important implications for the selection of HIV vaccines for expanded human trials.
[Show abstract][Hide abstract] ABSTRACT: CD8(+) "cytotoxic" T cells are important for the immune control of HIV and the closely related simian models SIV and chimeric simian-human immunodeficiency virus (SHIV), although the mechanisms of this control are unclear. One effect of CD8(+) T cell-mediated recognition of virus-infected cells is the rapid selection of escape mutant (EM) virus that is not recognized. To investigate the mechanisms of virus-specific CD8(+) T cell control during immune escape in vivo, we used a real-time PCR assay to study the dynamics of immune escape in early SHIV infection of pigtail macaques. For immune escape mediated by cytolysis, we would expect that the death rate of wild type (WT) infected cells should be faster than that of EM-infected cells. In addition, escape should be fastest during periods when the total viral load is declining. However, we find that there is no significant difference in the rate of decay of WT virus compared with EM virus. Further, immune escape is often fastest during periods of viral growth, rather than viral decline. These dynamics are consistent with an epitope-specific, MHC class I-restricted, noncytolytic mechanism of CD8(+) T cell control of SHIV that specifically inhibits the growth of WT virus in vivo.
Full-text · Article · Sep 2010 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: We developed highly expressing clade B and AE DNA and envelope protein (Env) vaccines for evaluation in mice and macaques as DNA prime/protein boost regimens. High levels of Env-specific antibodies were induced in mice, albeit with limited neutralizing activity in vitro. A combined clade B and AE regimen induced high titer Env-specific antibody in two pigtail macaques that neutralized several strains of HIV-1. However, upon mucosal challenge with SHIV(SF162P3) no protection from infection was observed. Although the vaccines tested provide a platform for inducing robust humoral immunity, further refinements to broaden coverage against divergent strains and induce mucosal immunity are needed.
[Show abstract][Hide abstract] ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is subject to both neutralizing antibody (NAb) and CD8
T-cell (cytotoxic T-lymphocyte [CTL]) immune pressure. We studied the reversion of the Env CTL escape mutant virus to the
wild type and the relationship between the reversion of CTL mutations with N-linked glycosylation site (NLGS)-driven NAb escape
in pigtailed macaques. Env CTL mutations either did not revert to the wild type or only transiently reverted 5 to 7 weeks
after infection. The CTL escape mutant reversion was coincident, for the same viral clones, with the loss of NLGS mutations.
At one site studied, both CTL and NLGS mutations were needed to confer NAb escape. We conclude that CTL and NAb escape within
Env can be tightly linked, suggesting opportunities to induce effective multicomponent anti-Env immunity.
Full-text · Article · Jul 2009 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Simian AIDS has a variable time course and presentation making it difficult to define disease effects of progressive simian immunodeficiency virus (SIV) infection. We commonly observed thrombocytopenia (TCP) associated with progressive SIV infection of pigtail macaques (Macaca nemestrina). We therefore analyzed the relationship between platelet counts, viral load (VL), and CD4 T-cell levels in 44 unselected macaques with chronic SIV infection. Persistent TCP was observed in 70% of pigtail macaques infected with SIVmac251 for up to 77 weeks in the absence of clinically significant bleeding. The presence of TCP correlated with higher SIV plasma VLs and depressed total and memory CD4 T cells. TCP was more common in macaques requiring euthanasia for incipient AIDS than macaques that survived to the end of the studies, although VL and CD4 T-cell decline were stronger independent predictors of AIDS-free survival. There was however no clear correlation between the development of TCP and immune activation as measured by plasma soluble CD14. We conclude that TCP is a useful end point to analyze SIV studies in pigtail macaques.
Full-text · Article · Jun 2009 · JAIDS Journal of Acquired Immune Deficiency Syndromes
[Show abstract][Hide abstract] ABSTRACT: There is an urgent need for human immunodeficiency virus (HIV) vaccines that induce robust mucosal immunity. Influenza A viruses (both H1N1 and H3N2) were engineered to express simian immunodeficiency virus (SIV) CD8 T-cell epitopes and evaluated following administration to the respiratory tracts of 11 pigtail macaques. Influenza virus was readily detected from respiratory tract secretions, although the infections were asymptomatic. Animals seroconverted to influenza virus and generated CD8 and CD4 T-cell responses to influenza virus proteins. SIV-specific CD8 T-cell responses bearing the mucosal homing marker beta7 integrin were induced by vaccination of naïve animals. Further, SIV-specific CD8 T-cell responses could be boosted by recombinant influenza virus-SIV vaccination of animals with already-established SIV infection. Sequential vaccination with influenza virus-SIV recombinants of different subtypes (H1N1 followed by H3N2 or vice versa) produced only a limited boost in immunity, probably reflecting T-cell immunity to conserved internal proteins of influenza A virus. SIV challenge of macaques vaccinated with an influenza virus expressing a single SIV CD8 T cell resulted in a large anamnestic recall CD8 T-cell response, but immune escape rapidly ensued and there was no impact on chronic SIV viremia. Although our results suggest that influenza virus-HIV vaccines hold promise for the induction of mucosal immunity to HIV, broader antigen cover will be needed to limit cytotoxic T-lymphocyte escape.
Full-text · Article · Jun 2009 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Escape mutant (EM) virus that evades CD8+ T cell recognition is frequently observed following infection with HIV-1 or SIV. This EM virus is often less replicatively "fit" compared to wild-type (WT) virus, as demonstrated by reversion to WT upon transmission of HIV to a naïve host and the association of EM virus with lower viral load in vivo in HIV-1 infection. The rate and timing of reversion is, however, highly variable. We quantified reversion to WT of a series of SIV and SHIV viruses containing minor amounts of WT virus in pigtail macaques using a sensitive PCR assay. Infection with mixes of EM and WT virus containing > or =10% WT virus results in immediate and rapid outgrowth of WT virus at SIV Gag CD8 T cell epitopes within 7 days of infection of pigtail macaques with SHIV or SIV. In contrast, infection with biologically passaged SHIV(mn229) viruses with much smaller proportions of WT sequence, or a molecular clone of pure EM SIV(mac239), demonstrated a delayed or slow pattern of reversion. WT virus was not detectable until > or =8 days after inoculation and took > or =8 weeks to become the dominant quasispecies. A delayed pattern of reversion was associated with significantly lower viral loads. The diversity of the infecting inoculum determines the timing of reversion to WT virus, which in turn predicts the outcome of infection. The delay in reversion of fitness-reducing CD8 T cell escape mutations in some scenarios suggests opportunities to reduce the pathogenicity of HIV during very early infection.
[Show abstract][Hide abstract] ABSTRACT: Simple and effective delivery methods for cellular immunotherapies are needed. We recently published on the effectiveness of using ex vivo pulsing of overlapping SIV Gag 15mer peptides onto fresh peripheral blood cells in 32 SIV(mac251)-infected pigtail macaques.
We now report on the safety of this approach, analysis of a novel assay for immunogenicity, the effect of an MHC allele, Mane-A*10, on CD8 T cell escape occurring and disease outcome.
The vaccine strategy was safe, with no perturbations in weight or hematological profiles in comparison to controls. The high levels of SIV-specific T cell immunogenicity of this approach was confirmed using a novel assay measuring upregulation of surface CD134 of CD4 T cells. A substantial effect of the Mane-A*10 allele in reducing SIV viral load of pigtail macaques was observed in both vaccinees and controls; the virologic efficacy of the immunotherapy in comparison to controls was greatest in Mane-A*10- animals. Escape mutations at several new CD8 T cell epitopes throughout the SIV proteome were observed, primarily in animals with poorer virologic control.
In summary, we provide further information that peptide-pulsed PBMC are a safe, immunogenic and effective immunotherapy. The observed influence of MHC alleles and immune escape allows us to design more insightful future immunotherapy studies.
Full-text · Article · Jan 2009 · Journal of Medical Primatology