In this manuscript, we provide in vivo preclinical evidence supporting the hypothesis that systemic antiretroviral PrEP can provide broad protection from HIV transmission. Our results obtained using a highly relevant in vivo model of HIV transmission show that systemic antiretroviral PrEP can effectively prevent rectal and intravenous HIV-1 infection. It is important to note that systemic antiretroviral PrEP with a single drug combination prevents infection of BLT mice by the three most common routes of human HIV-1 transmission. The highly encouraging results from this comprehensive evaluation of antiretroviral PrEP efficacy serve as strong proof of principle for this approach and have major implications for the continued planning and implementation of future and current PrEP studies.
Approaches aimed at obtaining protection from all potential modes of transmission are highly significant. Individually, unprotected vaginal intercourse accounts for the vast majority of new HIV transmissions globally 
. Rectal HIV exposure accounts for the majority of HIV transmission events in the United States and other developed nations 
. Even though rectal HIV exposure is the main mode of transmission among men who have sex with men, this route is also likely to account for a significant number of transmissions to women 
. Intravenous HIV exposure occurs primarily among injection drug users and is a growing health concern in many nations 
. Unlike mucosal exposure with its intrinsic physical and biological barriers, the direct exposure of virus to the blood stream results in more efficient transmission. Our results provide clear pre-clinical evidence of the potential usefulness of systemic PrEP for intravenous transmission. The ability to prevent HIV-1 transmission by all three routes using one drug combination has the potential of greatly facilitating the global implementation of preventative measures.
Until this study, in vivo
preclinical data substantiating a broad prevention approach using a single drug combination to prevent three routes of transmission had been lacking. In vivo
data on the efficacy of PrEP with FTC/TDF had been limited to two reports relating to mucosal transmission. In one study, we showed that systemic PrEP with FTC/TDF can effectively prevent vaginal HIV-1 transmission in BLT mice 
. The second study used rhesus macaques to show that intermittent or daily systemic PrEP with FTC/TDF can protect from rectal SHIV transmission in a low-dose repeat exposure model 
. Collectively, these two reports and the current data show that antiretroviral PrEP with FTC/TDF can afford extensive protection from vaginal, rectal and intravenous HIV-1 transmission.
When considering such broad use of antiretrovirals as prophylaxis, there is an issue of major importance that must be addressed. In humans, lack of strict compliance to PrEP regimens could increase the likelihood of drug resistance being developed in the event of breakthrough infection. Therefore broad antiretroviral use can result in increased emergence of resistance to the drug(s) when infections do occur 
. Spread of resistant viruses could limit the efficacy of current therapeutic interventions using these same drugs, although it should be noted that the fitness of multidrug resistant viruses for mucosal transmission has yet to be fully established. Future BLT mouse studies could model lack of compliance to evaluate the fitness of multidrug resistant viruses for mucosal transmission and explore potential mechanisms of breakthrough infections. Despite the high protection observed while using PrEP (>88%), our results indicated one breakthrough infection observed in one animal infected intravenously. It should be noted that sequence analysis of the entire reverse transcriptase gene revealed that this one transmission event was not the direct result of the appearance of mutations associated with drug-resistance 
. The molecular basis for transmission of wild type virus after venous exposure in the presence of PrEP remains to be determined.
Results obtained using humanized BLT mice must be considered in the context of previous studies of antiretrovirals for HIV prevention performed in other models such as non-human primates. Experiments performed using non-human primates have provided evidence for the use of tenofovir (PMPA) to prevent intravenous infection by SIVmne
in long-tailed macaques 
and successful antiretroviral PrEP in rhesus macaques exposed rectally to either SIVmac251/32H
have also been reported with this compound 
. Topical and systemic PrEP with one or more fusion inhibitors protected from vaginal SHIV transmission in rhesus macaques 
. Systemic PrEP with FTC/TDF was shown to prevent rectal SHIV transmission in rhesus macaques 
and in yet another non-human primate model, 2 pig-tailed macaques were protected from intravenous challenge with simian-tropic HIV (stHIV) by systemic PrEP with efavirenz plus FTC/TDF 
. Additional preclinical studies in macaques testing antiretroviral HIV-1 prevention modalities have focused on post-exposure prophylaxis, not pre-exposure regimens 
. The use of multiple animal models and different classes/combinations of drugs in these studies makes it difficult to make direct comparisons and to extrapolate potential outcomes. The current study represents a significant advance because it has produced a data set that can be easily interpreted and easily compared across multiple virus transmission routes all within the same experimental platform.
While our findings and those from non-human primate research suggest that antiretroviral PrEP can prevent HIV transmission, neither model has been shown to predict efficacy or safety in humans. This limitation exists because there is still no evidence of efficacy for antiretrovirals in preventing vaginal, rectal or intravenous transmission in humans 
. It will be essential that ongoing human clinical trial data be compared to BLT and non-human primate studies in order to validate these useful models. Protection is likely to be dependent on the drug exposure levels achieved following dosing of the PrEP antiretrovirals. Currently, there is no comparative pharmacological data of these levels between humans and BLT mice. Since protection is dependent on the dose of FTC/TDF it will be important to define the drug exposure of the regimen in BLT mice, and assess its relationship to the drug exposure achieved after oral dosing with Truvada in humans. Detailed information on the drug exposure in BLT mice will be important for interpreting the efficacy results in this model and for comparison with efficacy data from human trials when these become available in the near future. These data might help assess the relationship between drug exposure achieved after oral dosing with Truvada in humans and its effectiveness in ongoing clinical trials. In addition, a significant strength of humanized BLT mice is the fact that they can be used in future studies to address other potential variables between BLT mice and humans including differences in timing of dosing, drug concentrations, adherence to drug regimens, virus inoculum and relevant co-infections. Our results also suggest that humanized BLT mice will be useful for the evaluation of topical microbicides and to provide preclinical evidence for their potential success. The availability of a small animal model such as BLT mice for screening prevention modalities prior to or in conjunction with macaque and human studies is a great asset to the field 
In conclusion, we provide preclinical evidence regarding the potential efficacy of an antiretroviral pre-exposure prophylactic approach to prevent vaginal, rectal and intravenous HIV-1 transmission. Our results provide strong support for the continued implementation of clinical trials using targeted antiretroviral pre-exposure prophylaxis for all the major routes of HIV transmission contributing to the HIV/AIDS pandemic.