Larissa H. Haut

Capsid-specific T-cell Responses to Natural Infections With Adeno-associated Viruses in Humans Differ From Those of Nonhuman Primates

Hepatic adeno-associated virus serotype 2 (AAV2)-mediated gene transfer failed to achieve sustained transgene product expression in human subjects. We formulated the hypothesis that rejection of AAV-transduced hepatocytes is caused by AAV capsid-specific CD8(+) T cells that become reactivated upon gene transfer. Although this hypothesis was compatible with clinical data, which showed a rise in circulating AAV capsid-specific T cells following injection of AAV vectors, it did not explain that AAV vectors achieved long-term transgene expression in rhesus macaques, which are naturally infected with AAV serotypes closely related to those of humans. To address this apparent contradiction, we tested human and rhesus macaque samples for AAV capsid-specific T cells by intracellular cytokine staining combined with staining for T-cell subset and differentiation markers. This highly sensitive method, which could provide a tool to monitor adverse T-cell responses in gene transfer trials, showed that AAV capsid-specific CD8(+) and CD4(+) T cells can be detected in blood of naturally infected humans and rhesus macaques. They are present at higher frequencies in rhesus macaques. Furthermore, T cells from humans and rhesus macaques exhibit striking differences in their differentiation status and in their functions, which may explain the disparate duration of AAV-mediated gene transfer in these two species.

Augmentation of Primary Influenza A Virus-Specific CD8+ T Cell Responses in Aged Mice through Blockade of an Immunoinhibitory Pathway

Immune responses diminish with age resulting in an increased susceptibility of the elderly to infectious agents and an inability to mount protective immune responses to vaccines. Immunosenescence affects multiple aspects of the immune system, including CD8+ T cells, which control viral infections and are assumed to prevent the development of cancers. In this study, we tested if CD8+ T cell responses in aged mice could be enhanced through a vaccine that concomitantly expresses Ag and a molecule that blocks an immunoinhibitory pathway. Specifically, we tested a vaccine based on a replication-defective chimpanzee-derived adenovirus vector expressing the nucleoprotein (NP) of influenza A virus as a fusion protein with the HSV type 1 glycoprotein D, which through binding to the herpes virus entry mediator, blocks the immunoinhibitory herpes virus entry mediator B and T lymphocyte attenuator/CD160 pathways. Our results show that the vaccine expressing a fusion protein of NP and glycoprotein D induces significantly higher NP-specific CD8+ T cell responses in young and aged mice compared with the vaccine expressing NP only.

Obstacles to the successful development of an efficacious T cell-inducing HIV-1 vaccine

An efficacious vaccine to HIV‐1 is direly needed to stem the global pandemic. Immunogens that elicit broadly cross‐neutralizing antibodies to HIV‐1 remain elusive, and thus, most HIV‐1 vaccine efforts are focusing on induction of T cells. The notion that T cells can mediate protection against HIV‐1 has been called into question by the failure of the STEP trial, which was designed to test this concept by the use of an E1‐deleted Ad vaccine carrier. Lack of efficacy of the STEP trial vaccine underscores our limited knowledge about correlates of immune protection against HIV‐1 and stresses the need for an enhanced commitment to basic research, including preclinical and clinical vaccine studies. In this review, we discuss known correlates of protection against HIV‐1 and different vaccine strategies that have been or are being explored to induce such correlates, focusing on T cell‐inducing vaccines and particularly on Ad vectors.

Robust genital gag-specific CD8+ T-cell responses in mice upon intramuscular immunization with simian adenoviral vectors expressing HIV-1-gag

Most studies on E1‐deleted adenovirus (Ad) vectors as vaccine carriers for antigens of HIV‐1 have focused on induction of central immune responses, although stimulation of mucosal immunity at the genital tract (GT), the primary port of entry of HIV‐1, would also be highly desirable. In this study, different immunization protocols using chimpanzee‐derived adenoviral (AdC) vectors expressing Gag of HIV‐1 clade B given in heterologous prime‐boost regimens were tested for induction of systemic and genital immune responses. Although i.n. immunization stimulated CD8+ T‐cell responses that could be detected in the GT, this route induced only marginal cellular responses in systemic tissues and furthermore numbers of Gag‐specific CD8+ T cells contracted sharply within a few weeks. On the contrary, i.m. immunization induced higher and more sustained frequencies of vaccine‐induced cells which could be detected in the GT as well as systemic compartments. Antigen‐specific CD8+ T cells could be detected 1 year after immunization in all compartments analyzed. Genital memory cells secreted IFN‐γ, expressed high levels of CD103 and their phenotypes were consistent with a state of activation. Taken together, the results presented here show that i.m. vaccination with chimpanzee‐derived (simian) adenovirus vectors is a suitable strategy to induce a long‐lived genital CD8+ T‐cell response.

Vaccine-induced T cells Provide Partial Protection Against High-dose Rectal SIVmac239 Challenge of Rhesus Macaques

Despite enormous efforts by the scientific community, an effective HIV vaccine remains elusive. To further address to what degree T cells in absence of antibodies may protect against simian immunodeficiency virus (SIV) disease progression, rhesus macaques were vaccinated intramuscularly with a chimpanzee-derived Ad vector (AdC) serotype 6 and then boosted intramuscularly with a serologically distinct AdC vector of serotype 7 both expressing Gag of SIVmac239. Animals were subsequently boosted intramuscularly with a modified vaccinia Ankara (MVA) virus expressing Gag and Tat of the homologous SIV before mucosal challenge with a high dose of SIVmac239 given rectally. Whereas vaccinated animals showed only a modest reduction of viral loads, their overall survival was improved, in association with a substantial protection from the loss of CD4(+) T cells. In addition, the two vaccinated Mamu-A*01(+) macaques controlled viral loads to levels below detection within weeks after challenge. These data strongly suggest that T cells, while unable to affect SIV acquisition upon high-dose rectal infection, can reduce disease progression. Induction of potent T-cell responses should thus remain a component of our efforts to develop an efficacious vaccine to HIV-1.

Effect of Preexisting Immunity to Adenovirus on Transgene Product–Specific Genital T Cell Responses on Vaccination of Mice With a Homologous Vector

We evaluated changes in global and human immunodeficiency virus (HIV)-specific genital T cells after vaccination of female mice with a replication-defective adenovirus vector of human serotype 5 (AdHu5) expressing Gag protein of HIV-1, in the presence or absence of preexisting immunity to the vaccine carrier. Our data show that preexisting immunity causes a rapid and transient decrease of genital CD4(+) T cells without increasing the expression of chemokine (C-C motif) receptor 5. Furthermore, preexposure to AdHu5 affects long-term alterations in the magnitude and quality of vaccine-induced Gag-specific CD8(+) T cell responses. AdHu5-specific antibodies interfere with the induction of transgene product-specific CD8(+) T cell responses in systemic compartments, whereas some mechanism other than antibodies also seems to affect those that home to the genital tract.

Correlates of relative resistance against low-dose rectal simian immunodeficiency virus challenges in peripheral blood mononuclear cells of vaccinated rhesus macaques

In this study, we compared the immunogenicity and protection from repeated low‐dose intrarectal SIVmac251 challenge in two groups of vaccinated RMs. Animals were immunized with live SIVmac239, which had been attenuated by a deletion of the nef sequence, or they were vaccinated twice with an E1‐deleted AdHu5, expressing SIVmac239gag. The vaccinated animals and a cohort of unvaccinated control animals were then challenged 10 times in weekly intervals with low doses of SIVmac251 given rectally. Our results confirm previous studies showing that whereas SIVΔnef provides some degree of protection against viral acquisition after repeated low‐dose rectal SIVmac251 challenges, vaccination with an AdHu5gag vaccine designed to induce only antiviral T cell responses is ineffective. As immunological analyses of prechallenge, vaccine‐induced T and B cell responses failed to reveal correlates of protection that distinguished the more susceptible from the more resistant vaccinated animals, we carried out RNA‐Seq studies of paired pre‐ and postvaccination samples to identify transcriptional patterns that correlated with the differences in response. We show that gene expression signatures associated with the delayed SIV infection seen in some AdHu5gag recipients were largely present in prevaccination samples of those animals. In contrast, the responding SIVΔnef‐immunized animals showed a predominance of vaccine‐induced changes, thus enabling us to define inherited and vaccine‐induced gene expression signatures and their associated pathways that may play a role in preventing SIV acquisition.

Enhancement of recombinant adenovirus vaccine-induced primary but not secondary systemic and mucosal immune responses by all-trans retinoic acid

Vaccination is an important tool for enhancing immune responses against mucosal pathogens. Intramuscularly administered adenovirus (Ad) vectors have been demonstrated to be strong inducers of both systemic and mucosal immune responses. Further enhancement of immune responses following Ad vaccination is highly desirable. All-trans retinoic acid (ATRA), a biologically active vitamin A metabolite, has been explored as an adjuvant for primary immune responses following vaccination. In this study, we investigated the effect of ATRA on a heterologous Ad prime boost regimen. ATRA co-administration during priming increased mucosal and systemic antibody responses as well as mucosal but not systemic CD8(+) T cell responses. However, this effect was no longer apparent after boosting regardless of whether ATRA was administered at the time of priming, at the time of boosting, or at both immunizations. Our findings confirm ATRA as an adjuvant for primary immune responses and suggest that the adjuvant effect does not extend to secondary immune responses.

The effect of timing of influenza vaccination and sample collection on antibody titers and responses in the aged

Antibody responses, B cell subset distribution in blood and the blood transcriptome were analyzed in younger and aged human subjects before and after vaccination with the inactivated influenza vaccine. In the aged, but not the younger, individuals we saw a clear difference in antibody titers including those at baseline depending on the time of vaccination and sample collection. Differences in baseline titers in aged individuals treated in the morning or afternoon in turn affected responsiveness to the vaccine. In both younger and aged individuals, the time of sample collection also affected relative numbers of some of the B cell subsets in blood. A global gene expression analysis with whole blood samples from the aged showed small but statistically significant differences depending on the time of sample collection. Our data do not indicate that timing of vaccination affects immune responsiveness of the aged, but rather shows that in clinical influenza vaccine trials timing of collection of samples can have a major and potentially misleading influence on study outcome. In future vaccine trials, timing of vaccination and sample collection should be recorded carefully to allow for its use as a study covariant.

Race-related differences in antibody responses to the inactivated influenza vaccine are linked to distinct pre-vaccination gene expression profiles in blood

We conducted a 5-year study analyzing antibody and B cell responses to the influenza A virus components of the inactivated influenza vaccine, trivalent (IIV3) or quadrivalent (IIV4) in younger (aged 35-45) and aged (≥65 years of age) Caucasian and African American individuals. Antibody titers to the two influenza A virus strains, distribution of circulating B cell subsets and the blood transcriptome were tested at baseline and after vaccination while expression of immunoregulatory markers on B cells were analyzed at baseline. African Americans mounted higher virus neutralizing and IgG antibody responses to the H1N1 component of IIV3 or 4 compared to Caucasians. African Americans had higher levels of circulating B cell subsets compared to Caucasians. Expression of two co-regulators, i.e., programmed death (PD)-1 and the B and T cell attenuator (BTLA) were differentially expressed in the two cohorts. Race-related differences were caused by samples from younger African Americans, while results obtained with samples of aged African Americans were similar to those of aged Caucasians. Gene expression profiling by Illumina arrays revealed highly significant differences in 1368 probes at baseline between Caucasians and African Americans although samples from both cohorts showed comparable changes in transcriptome following vaccination. Genes differently expressed between samples from African Americans and Caucasians regardless of age were enriched for myeloid genes, while the transcripts that differed in expression between younger African Americans and younger Caucasians were enriched for those specific for B-cells.