Follicular helper CD4 T cells, T, residing in B-cell follicles within secondary lymphoid tissues, are readily infected by AIDS viruses and are a major source of persistent virus despite relative control of viral replication. This persistence is due at least in part to a relative exclusion of effective antiviral CD8 T cells from B-cell follicles. To determine whether CD8 T cells could be engineered to enter B-cell follicles, we genetically modified unselected CD8 T cells to express CXC chemokine receptor 5 (CXCR5), the chemokine receptor implicated in cellular entry into B-cell follicles. Engineered CD8 T cells expressing human CXCR5 (CD8) exhibited ligand-specific signaling and chemotaxis Six infected rhesus macaques were infused with differentially fluorescent dye-labeled autologous CD8 and untransduced CD8 T cells and necropsied 48 h later. Flow cytometry of both spleen and lymph node samples revealed higher frequencies of CD8 than untransduced cells, consistent with preferential trafficking to B-cell follicle-containing tissues. Confocal fluorescence microscopy of thin-sectioned lymphoid tissues demonstrated strong preferential localization of CD8 T cells within B-cell follicles with only rare cells in extrafollicular locations. CD8 T cells were present throughout the follicles with some observed near infected T In contrast, untransduced CD8 T cells were found in the extrafollicular T-cell zone. Our ability to direct localization of unselected CD8 T cells into B-cell follicles using CXCR5 expression provides a strategy to place highly effective virus-specific CD8 T cells into these AIDS virus sanctuaries and potentially suppress residual viral replication. AIDS virus persistence in individuals under effective drug therapy or those who spontaneously control viremia remains an obstacle to definitive treatment. Infected follicular helper CD4 T cells, T, present inside B-cell follicles represent a major source of this residual virus. While effective CD8 T-cell responses can control viral replication in conjunction with drug therapy or in rare cases spontaneously, most antiviral CD8 T cells do not enter B-cell follicles, and those that do fail to robustly control viral replication in the T population. Thus, these sites are a sanctuary and a reservoir for replicating AIDS viruses. Here, we demonstrate that engineering unselected CD8 T cells to express CXCR5, a chemokine receptor on T associated with B-cell follicle localization, redirects them into B-cell follicles. These proof of principle results open a pathway for directing engineered antiviral T cells into these viral sanctuaries to help eliminate this source of persistent virus.

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AIDS virus infections are rarely controlled by cell-mediated immunity, in part due to viral immune evasion and immunodeficiency resulting from CD4؉ T-cell infection. One likely aspect of this failure is that antiviral cellular immune responses are either absent or present at low levels during the initial establishment of infection. To test whether an extensive, timely, and effective response could reduce the establishment of infection from a high-dose inoculum, we adoptively transferred large numbers of T cells that were molecularly engineered with anti-simian immunodeficiency virus (anti-SIV) activity into rhesus macaques 3 days following an intrarectal SIV inoculation. To measure in vivo antiviral activity, we assessed the number of viruses transmitted using SIVmac239X, a molecularly tagged viral stock containing 10 genotypic variants, at a dose calculated to transmit 12 founder viruses. Single-genome sequencing of plasma virus revealed that the two animals receiving T cells expressing SIV-specific T-cell receptors (TCRs) had significantly fewer viral genotypes than the two control animals receiving non-SIV-specific T cells (means of 4.0 versus 7.5 transmitted viral genotypes; P ‫؍‬ 0.044). Accounting for the likelihood of transmission of multiple viruses of a particular genotype, the calculated means of the total number of founder viruses transmitted were 4.5 and 14.5 in the experimental and control groups, respectively (P ‫؍‬ 0.021). Thus, a large antiviral T-cell response timed with virus exposure can limit viral transmission. The presence of strong, preexisting T-cell responses, including those induced by vaccines, might help prevent the establishment of infection at the lower-exposure doses in humans that typically transmit only a single virus. IMPORTANCEThe establishment of AIDS virus infection in an individual is essentially a race between the spreading virus and host immune defenses. Cell-mediated immune responses induced by infection or vaccination are important contributors in limiting viral replication. However, in human immunodeficiency virus (HIV)/SIV infection, the virus usually wins the race, irreversibly crippling the immune system before an effective cellular immune response is developed and active. We found that providing an accelerated response by adoptively transferring large numbers of antiviral T cells shortly after a high-dose mucosal inoculation, while not preventing infection altogether, limited the number of individual viruses transmitted. Thus, the presence of strong, preexisting T-cell responses, including those induced by vaccines, might prevent infection in humans, where the virus exposure is considerably lower. T he CD8ϩ T cell plays an important role in control of AIDS viruses, i.e., human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) (1-10). However, in most cases, CD8 ϩ T-cell responses, whether consisting of responses to infection developed de novo or to prior vaccination, are unable to prevent development of persistent, disseminated infection....

Mucosal surfaces are the predominant site of human immunodeficiency virus (HIV)-1 transmission. For prophylactic approaches to effectively prevent HIV infection and subsequent dissemination, the induction of mucosally relevant protective immunity will be critical. Here, we have characterized the antibody (Ab) response generated by a highly conserved gp41epitope, QARVLAVERY, in an optimized immunization model that elicits potent epitope-specific Abs in the serum, vaginal washes, and fecal secretions of immunized mice. Our results show that QARVLAVERY is indeed a potent inducer of IgA and importantly, QARVLAVERY-specific IgA was effective in neutralizing HIV and inhibiting viral transcytosis. Intriguingly, QARVLAVERY also generated an approximate 1:1 ratio of IgG:IgA in the serum of immunized mice, independent of the delivery regimen and produced early systemic IgA, even before IgG. In light of the significantly high IgA induction by QARVLAVERY and the functionality of epitope-specific Abs in the inhibition of HIV infection and transcytosis, QARVLAVERY is an attractive epitope to be considered in mucosal vaccination strategies against HIV.

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Here, we present an improved strategy for producing T-cell receptor (TCR)-expressing retroviral vectors using a “Golden Gate” cloning strategy. This method takes advantage of the modular nature of TCR genes by directly amplifying TCR α and β variable regions from RNA or cDNA, then cloning and fusing them with their respective constant region genes resident in a retroviral TCR expression vector. This one step approach greatly streamlines the TCR vector production process versus the traditional three-step process that typically involves cloning whole TCR genes, producing a TCR expression cassette, and constructing a retroviral construct. To date, we have produced TCR vectors which transferred 7 functional human/rhesus macaque TCRs into primary T cells for studies using this method. This approach has promise to assemble other genes with defined variable regions such as immunoglobulins.

To study CD4+ T-cell suppression of AIDS virus replication, we isolated nine rhesus macaque SIVGag-specific CD4+ T-cell clones. One responding clone, Gag68, produced a typical cytotoxic CD8+ T-cell response: induction of intracellular IFN-γ, MIP-1α, MIP-1β, and CD107a degranulation. Gag68 effectively suppressed the spread of SIVmac239 in CD4+ T cells with a corresponding reduction of infected Gag68 effector cells, suggesting that CD4+ effectors need to suppress their own infection in addition to their targets to be effective. Gag68 TCR cloning and gene transfer into CD4+ T cells enabled additional experiments with this unique specificity after the original clone senesced. Our data supports the idea that CD4+ T cells can directly limit AIDS virus spread in T cells. Furthermore, Gag68 TCR transfer into CD4+ T-cell clones with differing properties holds promise to better understand the suppressive effector mechanisms used by this important component of the antiviral response using the rhesus macaque model.

BackgroundThe TRIM5α protein is a principal restriction factor that contributes to an HIV-1 replication block in rhesus macaque CD4+ T cells by preventing reverse transcription. HIV-1 restriction is induced in human CD4+ T cells by expression of rhesus TRIM5α as well as those of other old world monkeys. While TRIM5α restriction has been extensively studied in single-round infection assays, fewer studies have examined restriction after extended viral replication.ResultsTo examine TRIM5α restriction of replication, we studied the ability of TRIM5α proteins from African green monkey (AgmTRIM5α) and gorilla (gorTRIM5α) to restrict HIV-1 and SIVmac239 replication. These xenogeneic TRIM5α genes were transduced into human Jurkat-CCR5 cells (JR5), which were then exposed to HIV-1 or SIVmac239. In our single-round infection assays, AgmTRIM5α showed a relatively modest 4- to 10-fold restriction of HIV-1 and SIVmac239, while gorTRIM5α produced a 2- and 3-fold restriction of HIV-1 and SIVmac239, respectively, consistent with the majority of previously published single-round studies. To assess the impact of these modest effects on infection, we tested restriction in replication systems initiated with either cell-free or cell-to-cell challenges. AgmTRIM5α powerfully restricted both HIV-1 and SIVmac239 replication 14 days after cell-free infection, with a ≥ 3-log effect. Moreover, expression of AgmTRIM5α restricted HIV-1 and SIVmac239 replication by 2-logs when co-cultured with infected JR5 cells for 12 days. In contrast, neither expression of gorTRIM5α nor rhesus TRIM5α induced significant resistance when co-cultured with infected cells. Follow up experiments showed that the observed differences between replication and infection were not due to assembly defects as xenogeneic TRIM5α expression had no effect on either virion production or specific infectivity.ConclusionsOur results indicate that AgmTRIM5α has a much greater effect on extended replication than on any single infection event, suggesting that AgmTRIM5α restriction acts cumulatively, building up over many rounds of replication. Furthermore, AgmTRIM5α was able to potently restrict both HIV-1 and SIV replication in a cell-to-cell infection challenge. Thus, AgmTRIM5α is unique among the TRIM5α species tested to date, being able to restrict even at the high multiplicities of infection presented by mixed culture with nonrestrictive infected cells.