We show that PrEP with anti-retrovirals can facilitate the development of adaptive T cell immunity in response to mucosal virus exposures. This “chemo-vaccination” effect was documented in detail by analyzing incidence, duration, epitope specificities, functional properties, differentiation markers, and protective effects of T cells. This confirms earlier chemo-vaccination reports either during pre-exposure prophylaxis 
, or following effective anti-retroviral treatment begun shortly after inoculation 
. We significantly expanded on earlier studies by using a cut-off for positive anti-viral T cell responses based on background measurements in naïve animals. This allowed us to demonstrate that chemo-vaccination-induced T cell responses are not merely pre-existing background responses with cross-reactivity to SHIV-derived antigens.
Chemo-vaccination induced anti-SHIV T cells in two of four PrEP-protected macaques, and in one of two macaques before they failed PrEP. One reason for the incomplete response rates could be variations in viral antigen presentation in individual macaques, e.g., due to MHC allelic variability. It is noteworthy that macaque 35451 with strong T cell responses was the only study macaque harboring Mamu-B08, an allele associated with superior control of SIV 
. The other macaque with chemo-vaccination-induced T cells (4284), on the other hand, was not unique in its genetic MHC make-up. It is possible that T cell responses developed, but were not detected in all macaques, due to different induction kinetics, trafficking behavior, or compartmentalization.
A major limitation was the small number of animals at the beginning of the study, and further subdivision of animals due to different experimental outcomes. Only one animal with anti-SHIV T cells could be re-exposed to virus, and only when its T cell responses had subsided. Nevertheless, the in-depth description of chemo-vaccination and long follow-up time in this study provide useful information for future study designs to further examine incidence and consequences of chemo-vaccination in macaques and in limited samples from human clinical PrEP trials. In future studies, it would also be worthwhile to comprehensively evaluate mucosal specimens, and tissue- or lymphatic tissue-resident lymphocytes.
We characterized chemo-vaccination induced T cells. Life-span of the cells was limited to less than 27 weeks, while memory T cells induced by potent vaccines or infection can last for years if not a lifetime 
. Re-stimulation of previously expanded T cells appeared impaired, because dominant epitope specificities changed rapidly. This suggests that immune memory qualities were not well developed. Memory T cells were mostly of TCM
phenotype, while superior protection from and control of SIV by mucosal TEM
cells has been reported 
. Lastly, we noticed a focus on pol
epitopes, rather than env
immune-reactivity can be effective 
, but efficient viral control is better documented for gag
–directed T cells 
. It is noteworthy that a study in HIV+
individuals exposed to other HIV strains by their HIV+
partners, but not super-infected, also found T cells focused on pol
. In addition, dominant T cell responses to pol
rather than env
have been reported for EU individuals 
. Focus on pol
might therefore be a feature of T cells elicited by priming in the absence of productive infection. It is possible that different tests for T cell differentiation and function (e.g., for proliferative or cytotoxic capabilities) would have revealed additional T cell characteristics, but no additional specimens were available for such analyses.
The high frequency of virus exposures likely prevented efficient functional T cell memory maturation and survival. It is important to emphasize, however, that the study was not designed to optimally induce T cell immunity. Rather, we addressed what kind of immunity is induced by physiologic virus exposures concurrent with partially effective PrEP. Thus, we consider our results relevant for the type of T cell immunity that can be expected following an intermittent PrEP regimen in sexually active people.
Having a history of SHIV-specific T cells due to chemo-vaccination did not protect macaque 4284 from SHIV infection, nor did it delay or significantly accelerate its infection. Ensuing viremia was remarkably similar in another re-challenged macaque (33246), although 33246 had never shown anti-SHIV T cell responses. Both macaques were identical at eight MHC alleles known to impact viral control. Limitations of these studies include: Only one animal was tested, and re-exposure occurred when peripheral blood virus-specific T cells had waned. Another macaque, 34912, became infected after 14 viral exposures, despite weak T cell responses after the 13th exposure. It is unclear whether the animal was already in the viral eclipse phase when T cells were detected. Further research is needed to determine a potentially increased or decreased infection risk after chemo-vaccination during subsequent virus exposures.
T cells matured further in macaque 35451 after resting and undergoing more chemo-vaccination, but it is not clear whether minor changes in epitope specificities and memory phenotype significantly affected anti-viral functions. Chemo-vaccination-induced T cell immunity could possibly be improved by combining PrEP with a vaccine targeted to induce specific immune pathways. A complex vaccine with an antibody-inducing component could complement the observed lack of antibodies; another component could accelerate limited TEM
differentiation. Deliberately skewing T cells towards TEM
phenotype would most likely also increase mucosal anti-viral responses 
. Moreover, the RV144 HIV vaccine trial suggested that vaccine efficacy may have decreased over the first year after vaccination 
. If a vaccine is administered prior to PrEP, chemo-vaccination could possibly boost and extend limited vaccine efficacy. Thus, combination of these two HIV prevention methods could have additive or synergistic effects and raise their combined efficacy significantly. These issues could be addressed by further research in nonhuman primate models employing comparative groups of single or combined prevention modalities, administered during controlled virus exposures.
Anti-SHIV T cells were composed of CD4+
cells, suggesting that antigen presentation likely occurred in MHC class I and II pathways. It is possible that antigen presentation was mostly the result of uptake of virus particles by resident antigen presenting cells through endocytic mechanisms. Many experimental SHIV stocks contain a large proportion of replication-defective, non-infectious, or severely attenuated virions; these may induce immune responses by this mechanism. We previously found no anti-SHIV T cell responses in macaques exposed rectally up to 14 times to virus without concurrent PrEP 
, although they have been observed after vaginal exposure 
. It is possible that PrEP-treated macaques experienced a low level of initial virus replication that remained local, and did not result in systemic infection due to the anti-retroviral action of Truvada. Because the PrEP regimen was partially effective, and did not block infection in all macaques, there may have been more transient/abortive infection than during highly effective PrEP, resulting in stronger induction of immune responses, as has been documented in earlier PrEP studies of perinatal SIV infection by Van Rompay et al 
. Even if initial local replication occurred, it likely did not persist in a suppressed state, because we did not detect it using three assays (detection of plasma viral RNA load, proviral DNA in PBMCs, and serum anti-SHIV antibodies) during extended follow-up (data not shown). In addition, we previously reported no evidence for occult infection in similarly PrEP-protected macaques after in-vivo depletion of CD8+
T cells 
It is not clear why chemo-vaccination induced no B cell immunity despite considerable T cell immunity. B cell induction requires CD4+
T cell help. High-avidity memory CD4+
T cells are induced by more prolonged antigenic stimulation than CD8+
. It is possible that chemo-vaccination-induced anti-SHIV CD4+
T cells did not differentiate enough to become efficient B cell helpers. Alternatively, viral antigens possibly did not reach lymphoid tissues necessary for B cell maturation 
. Mucosal secretions were not tested for the presence of anti-viral antibodies, and it is therefore possible that mucosal but not systemic antibodies were induced by chemo-vaccination. In addition, low-level SHIV-specific antibodies may have been below the limit of detection of the Bio-Rad 1/2 plus O EIA assay, although this assay is considered more sensitive than commercial Western Blotting techniques for the diagnosis of sero-conversion.
It is currently unclear whether human iPrEx 
or other PrEP trial participants experienced chemo-vaccination effects and whether this is impacted by HIV exposure frequency. Exposure interval effects have been observed in EU commercial sex workers, including a group who lost T cell responses after taking a 2-month or longer break from sex work 
. Potential protection associated with chemo-vaccination could be determined when individuals are retrospectively grouped into HIV-reactive and non-reactive PrEP participants. Given the variations in human behavior and HIV exposure, however, nonhuman primate models have great advantages for further study of the consequences of chemo-vaccination. Animals can be sampled very frequently; in contrast, in clinical trials, the larger intervals between visits make it more likely that chemo-vaccination effects are missed.
In conclusion, intermittent Truvada-based oral PrEP regimens can facilitate induction of T cell immunity following repeated virus exposure. Occurrence and implications of such chemo-vaccination effects should be evaluated in specimens from current human PrEP trials. We suggest that combination of PrEP and vaccines might be more efficacious than either intervention alone. Such studies can be informed by comprehensive examination of immune responses and outcomes of vaccines, PrEP, microbicides, and other combination approaches aimed at interrupting HIV transmission.