Doctor of Philosophy, Harvard University (2011)
Bachelor of Science, Brown University (2004)
John Boothroyd, Postdoctoral Faculty Sponsor
Mycobacterium bovis bacillus Calmette-Guérin (BCG) is an attractive target for development as a live vaccine vector delivering transgenic antigens from HIV and other pathogens. Most studies aimed at defining the clearance of BCG have been performed at doses between 10(2) and 10(4) CFU. Interestingly, however, recombinant BCG (rBCG) administered at doses of >10(6) CFU effectively generates antigen-specific T-cell responses and primes for heterologous boost responses. Thus, defining clearance at high doses might aid in the optimization of rBCG as a vector. In this study, we used bioluminescence imaging to examine the kinetics of rBCG transgene expression and clearance in mice immunized with 5 × 10(7) CFU rBCG expressing luciferase. Similar to studies using low-dose rBCG, our results demonstrate that the adaptive immune response is necessary for long-term control of rBCG beginning 9 days after immunizing mice. However, in contrast to these reports, we observed that the majority of mycobacterial antigen was eliminated prior to day 9. By examining knockout and antibody-mediated depletion mouse models, we demonstrate that the rapid clearance of rBCG occurs in the first 24 h and is mediated by Gr-1(+) cells. As Gr-1(+) granulocytes have been described as having no impact on BCG clearance at low doses, our results reveal an unappreciated role for Gr-1(+) neutrophils and inflammatory monocytes in the clearance of high-dose rBCG. This work demonstrates the potential of applying bioluminescence imaging to rBCG in order to gain an understanding of the immune response and increase the efficacy of rBCG as a vaccine vector.
View details for DOI 10.1128/CVI.00363-14
View details for PubMedID 24920602
Live-attenuated, non-pathogenic bacillus Calmette-Guérin (BCG) mediates long-lasting immune responses, has been safely administered as tuberculosis vaccine to billions of humans, and is affordable to produce as a vaccine vector. These characteristics make it very attractive as an HIV vaccine vector candidate. Here, we assessed the immunogenicity of recombinant BCG (rBCG) constructs with different SIVgag-expression cassettes as priming agents followed by a recombinant replication-incompetent NYVAC boost in rhesus macaques. Unmutated rBCG constructs were used in comparison to mutants with gene deletions identified in an in vitro screen for augmented immunogenicity. We demonstrate that BCG-SIVgag is able to elicit robust transgene-specific priming responses resulting in strong SIV epitope-specific cellular immune responses. While enhanced immunogenicity was sustained at moderate levels for over a year following the heterologous boost vaccination, we were unable to demonstrate a protective effect after repeated rectal mucosal challenges with pathogenic SIVmac251. Our findings highlight the potential for rBCG vaccines to stimulate effective cross-priming and enhanced major histocompatibility complex class I presentation, suggesting that combining this approach with other immunogens may contribute to development of effective vaccine regimens against HIV.
View details for DOI 10.1128/CVI.00324-14
View details for PubMedID 25080550
Although most research to date on Trace Amine Associated Receptor 1 (TAAR1) has focused on its role in the brain, it has been recognized since its discovery in 2001 that TAAR1 mRNA is expressed in peripheral tissues as well, suggesting that this receptor may play a role in non-neurological pathways. This study reports TAAR1 expression, signaling and functionality in rhesus monkey lymphocytes. We detected a high level of TAAR1 protein in immortalized rhesus monkey B cell lines and a significant upregulation of TAAR1 protein expression in rhesus monkey lymphocytes following PHA treatment. Through screening a wide range of signaling pathways for their upregulation following TAAR1 activation by its potent agonist methamphetamine, we identified two transcription factors, CREB and NFAT, which are commonly associated with immune activation. Furthermore, we observed a TAAR1-dependent phosphorylation of PKA and PKC following treatment with methamphetamine in transfected HEK293 cells, immortalized rhesus monkey B cells and PHA-activated rhesus monkey lymphocytes. Accordingly, the high levels of TAAR1 that we observed on lymphocytes are inducible and fully functional, capable of transmitting a signal likely via PKA and PKC activation following ligand binding. More importantly, an increase in TAAR1 receptor expression is concomitant with lymphocyte immune activation, suggesting a possible role for TAAR1 in the generation or regulation of an immune response. TAAR1 is emerging as a potential therapeutic target, with regard to its ability to modulate brain monoamines. The current data raises the possibility that TAAR1-targeted drugs may also alter immune function.
View details for DOI 10.1007/s11481-011-9321-4
View details for Web of Science ID 000312363500017
View details for PubMedID 22038157
While immunological memory has long been considered the province of T- and B-lymphocytes, it has recently been reported that innate cell populations are capable of mediating memory responses. We now show that an innate memory immune response is generated in mice following infection with vaccinia virus, a poxvirus for which no cognate germline-encoded receptor has been identified. This immune response results in viral clearance in the absence of classical adaptive T and B lymphocyte populations, and is mediated by a Thy1(+) subset of natural killer (NK) cells. We demonstrate that immune protection against infection from a lethal dose of virus can be adoptively transferred with memory hepatic Thy1(+) NK cells that were primed with live virus. Our results also indicate that, like classical immunological memory, stronger innate memory responses form in response to priming with live virus than a highly attenuated vector. These results demonstrate that a defined innate memory cell population alone can provide host protection against a lethal systemic infection through viral clearance.
View details for DOI 10.1371/journal.ppat.1002141
View details for Web of Science ID 000294298100004
View details for PubMedID 21829360
While it is well established that CD8(+) T cells generated in the absence of CD4(+) T cells mediate defective recall responses, the mechanism by which CD4(+) T cells confer help in the generation of CD8(+) T-cell responses remains poorly understood. To determine whether CD4(+) T-cell-derived IL-21 is an important regulator of CD8(+) T-cell responses in help-dependent and -independent viral infections, we examined these responses in the IL-21Rα(-/-) mouse model. We show that IL-21 has a role in primary CD8(+) T-cell responses and in recall CD8(+) T-cell responses in help-dependent viral infections. This effect is due to a direct action of IL-21 in enhancing the proliferation of virus-specific CD8(+) T cells and reducing their TRAIL expression. These findings indicate that IL-21 is an important mediator of CD4(+) T-cell help to CD8(+) T cells.
View details for DOI 10.1002/eji.200939939
View details for Web of Science ID 000284059000014
View details for PubMedID 21061439
In order to increase the immune breadth of human immunodeficiency virus (HIV) vaccines, strategies such as immunization with several HIV antigens or centralized immunogens have been examined. HIV-1 gp120 protein is a major immunogen of HIV and has been routinely considered for inclusion in both present and future AIDS vaccines. However, recent studies proposed that gp120 interferes with the generation of immune response to codelivered antigens. Here, we investigate whether coimmunization with plasmid-encoded gp120 alters the immune response to other coadministered plasmid encoded antigens such as luciferase or ovalbumin in a mouse model. We found that the presence of gp120 leads to a significant reduction in the expression level of the codelivered antigen in vivo. Antigen presentation by antigen-presenting cells was also reduced and resulted in the induction of weak antigen-specific cellular and humoral immune responses. Importantly, gp120-mediated immune interference was observed after administration of the plasmids at the same or at distinct locations. To characterize the region in gp120 mediating these effects, we used plasmid constructs encoding gp120 that lacks the V1V2 loops (DeltaV1V2) or the V3 loop (DeltaV3). After immunization, the DeltaV1V2, but not the DeltaV3 construct, was able to reduce antigen expression, antigen presentation, and subsequently the immunogenicity of the codelivered antigen. The V3 loop dependence of this phenomenon seems to be limited to V3 loops known to interact with the CXCR4 molecule but not with CCR5. Our study presents a novel mechanism by which HIV-1 gp120 interferes with the immune response against coadministered antigen in a polyvalent vaccine preparation.
View details for DOI 10.1128/JVI.00394-09
View details for Web of Science ID 000270602300009
View details for PubMedID 19692474
While recent studies have demonstrated that secondary CD8+ T cells develop into effector-memory cells, the impact of particular vaccine regimens on the elicitation of these cells remains poorly defined. In the present study we evaluated the effect of three different immunogens--recombinant vaccinia, recombinant adenovirus, and plasmid DNA--on the generation of memory cellular immune responses. We found that vectors that induce the rapid movement of CD8+ T cells into the memory compartment during a primary immune response also drive a rapid differentiation of these cells into effector-memory CD8+ T cells following a secondary immunization. In contrast, the functional profiles of both CD8+ and CD4+ T cells, assessed by measuring antigen-stimulated gamma interferon and interleukin-2 production, were not predominantly shaped by the boosting immunogen. We also demonstrated that the in vivo expression of antigen by recombinant vectors was brief following boosting immunization, suggesting that antigen persistence has a minimal impact on the differentiation of secondary CD8+ T cells. When used in heterologous or in homologous prime-boost combinations, these three vectors generated antigen-specific CD8+ T cells with different phenotypic profiles. Expression of the memory-associated molecule CD27 on effector CD8+ T cells decreased following heterologous but not homologous boosting, resulting in a phenotypic profile similar to that seen on primary CD8+ T cells. These data therefore suggest that the phenotype of secondary CD8+ T cells is determined predominantly by the boosting immunogen whereas the cytokine profile of these cells is shaped by both the priming and boosting immunogens.
View details for DOI 10.1128/JVI.01519-07
View details for Web of Science ID 000251330500009
View details for PubMedID 17881444
The duration of Ag expression in vivo has been reported to have a minimal impact on both the magnitude and kinetics of contraction of a pathogen-induced CD8(+) T cell response. In this study, we controlled the duration of Ag expression by excising the ear pinnae following intradermal ear pinnae DNA immunization. This resulted in decreased magnitude, accelerated contraction and differentiation, and surprisingly greater secondary CD8(+) T cell responses. Furthermore, we found delayed and prolonged Ag presentation in the immunized mice; however, this presentation was considerably decreased when the depot Ag was eliminated. These findings suggest that the magnitude and the contraction phase of the CD8(+) T cell response following intradermal DNA immunization is regulated by the duration rather than the initial exposure to Ag.
View details for Web of Science ID 000250792700040
View details for PubMedID 17982062
The most promising vaccine strategies for the induction of cytotoxic-T-lymphocyte responses have been heterologous prime/boost regimens employing a plasmid DNA prime and a live recombinant-vector boost. The priming immunogen in these regimens must elicit antigen-specific memory CD8+ T lymphocytes that will expand following the boosting immunization. Because plasmid DNA immunogens are expensive and their immunogenicity has proven disappointing in human clinical trials, we have been exploring novel priming immunogens that might be used in heterologous immunization regimens. Here we show that priming with a prototype recombinant Mycobacterium smegmatis strain expressing human immunodeficiency virus type 1 (HIV-1) gp120-elicited CD4+ T lymphocytes with a functional profile of helper cells as well as a CD8+ T-lymphocyte population. These CD8+ T lymphocytes rapidly differentiated to memory cells, defined on the basis of their cytokine profile and expression of CD62L and CD27. Moreover, these recombinant-mycobacterium-induced T lymphocytes rapidly expanded following boosting with a recombinant adenovirus expressing HIV-1 Env to gp120-specific CD8+ T lymphocytes. This work demonstrates a remarkable skewing of recombinant-mycobacterium-induced T lymphocytes to durable antigen-specific memory CD8+ T cells and suggests that such immunogens might be used as priming vectors in prime/boost vaccination regimens for the induction of cellular immune responses.
View details for DOI 10.1128/JVI.01269-06
View details for Web of Science ID 000242958600006
View details for PubMedID 17050608