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Human immunodeficiency virus (HIV) vaccines have not been successful in clinical trials. Dimeric IgA (dIgA) in the form of secretory IgA is the most abundant antibody class in mucosal tissues, making dIgA a prime candidate for potential HIV vaccines. We coupled Positron Emission Tomography (PET) imaging and fluorescent microscopy of 64Cu-labeled, photoactivatable-GFP HIV (PA-GFP-BaL) and fluorescently labeled dIgA to determine how dIgA antibodies influence virus interaction with mucosal barriers and viral penetration in colorectal tissue. Our results show that HIV virions rapidly disseminate throughout the colon two hours after exposure. The presence of dIgA resulted in an increase in virions and penetration depth in the transverse colon. Moreover, virions were found in the mesenteric lymph nodes two hours after viral exposure, and the presence of dIgA led to an increase in virions in mesenteric lymph nodes. Taken together, these technologies enable in vivo and in situ visualization of antibody-virus interactions and detailed investigations of early events in HIV infection., Author summary Vaccines provide protection in humans by eliciting the production of antibodies when exposed to a specific pathogen. Currently, an effective human immunodeficiency virus (HIV) vaccine does not exist. Since the beginning of the HIV epidemic, approximately 38 million people have died, creating the need to develop an HIV vaccine. The most common antibody in the organs that are exposed to HIV is dimeric IgA (dIgA). Here, we used multiple imaging techniques to determine how HIV travels throughout the colon once introduced into the body and how dIgA influences HIV movement in the rectum. We found that dIgA increased the amount of HIV found in the colon, the distance it travelled, and the depth into tissues that HIV penetrated. dIgA also increased the amount of HIV in the mesenteric lymph nodes two hours after viral exposure. Our study shows these imaging technologies can be used to examine interactions between viruses and antibodies in early HIV infection in the natural context of the anatomy and physiology of the rhesus macaque model.

Vertical transmission of human immunodeficiency virus (HIV) can occur in utero, during delivery, and through breastfeeding. We utilized Positron Emission Tomography (PET) imaging coupled with fluorescent microscopy of 64Cu-labeled photoactivatable-GFP-HIV (PA-GFP-BaL) to determine how HIV virions distribute and localize in neonatal rhesus macaques two and four hours after oral viral challenge. Our results show that by four hours after oral viral exposure, HIV virions localize to and penetrate the rectal mucosa. We also used a dual viral challenge with a non-replicative viral vector and a replication competent SHIV-1157ipd3N4 to examine viral transduction and dissemination at 96 hours. Our data show that while SHIV-1157ipd3N4 infection can be found in the oral cavity and upper gastrointestinal (GI) tract, the small and large intestine contained the largest number of infected cells. Moreover, we found that T cells were the biggest population of infected immune cells. Thus, thanks to these novel technologies, we are able to visualize and delineate of viral distribution and infection throughout the entire neonatal GI tract during acute viral infection., Author summary Approximately 1.8 million children are currently living with human immunodeficiency virus (HIV). While mother-to-child HIV transmission can occur in utero and during delivery, it most commonly occurs through breastfeeding, creating the need to understand how the virus moves throughout the body and infects the infant once breast milk is consumed. Here, we used multiple imaging techniques and PCR to determine how HIV distributes throughout the gastrointestinal tract after oral viral exposure and in which tissues and cell types become acutely infected. We found that HIV rapidly spreads throughout and penetrates the entire gastrointestinal tract as early as four hours after exposure. We also found that the intestine contained the largest number of infected cells at 96 hours and that most cells infected were T cells. Our study shows that these imaging technologies allow for the examination of viral distribution and infection in a rhesus macaque model.

Currently, there are mounting data suggesting that HIV-1 acquisition in women can be affected by the use of certain hormonal contraceptives. However, in non-human primate models, endogenous or exogenous progestin-dominant states are shown to increase acquisition. To gain mechanistic insights into this increased acquisition, we studied how mucosal barrier function and CD4+ T-cell and CD68+ macrophage density and localization changed in the presence of natural progestins or after injection with high-dose DMPA. The presence of natural or injected progestins increased virus penetration of the columnar epithelium and the infiltration of susceptible cells into a thinned squamous epithelium of the vaginal vault, increasing the likelihood of potential virus interactions with target cells. These data suggest that increasing either endogenous or exogenous progestin can alter female reproductive tract barrier properties and provide plausible mechanisms for increased HIV-1 acquisition risk in the presence of increased progestin levels., Author Summary Sexual transmission accounts for over 80% of all HIV-1 infections, with half of new infections occurring in women. Epidemiological studies suggest that certain hormonal contraceptives may be associated with increased HIV-1 acquisition. A hormonal influence of vaginal HIV acquisition is supported by studies utilizing various non-human primate models, which reveal that susceptibility to vaginal transmission is affected by the menstrual cycle or exogenous hormonal treatment. However, the mechanism of increased susceptibility remains unknown. Here, utilizing a variety of techniques and non-human primates, we illustrate that progestin-based contraceptives, such as Depo-Provera, and the natural menses luteal phase have a very similar impact in these models systems. Both increase virus influx into the endocervix and stimulate target cell infiltration of the squamous epithelium. The shedding of the superficial dead layers of the squamous epithelium in the progestin dominant state put the tissue resident cells in close proximity with virus in the lumen increasing the possibility of interactions and infection. A better understanding of vaginal SIV/SHIV acquisition in these widely utilized models is essential to interpret the results of ongoing clinical trials of HIV-1 acquisition during hormonal contraceptives use. An increased understanding of the mechanisms of HIV acquisition should contribute to vaccine development.

Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine-induced antibodies and therapeutic antibodies will enable a better understanding of their capacity to prevent and/or control HIV-1 infection in vivo., Author Summary Emerging data highlight the role of antibody Fc effector functions as immunological mechanisms involved in vaccine and passive immunotherapy efficacy. One such Fc effector function is antibody-mediated virion internalization, where antibodies recognize a virus and engage Fc receptors on phagocytes, causing them to internalize the virus. Although potentially critical for protection from HIV-1 acquisition, the ability of HIV-1 specific antibodies to mediate virion internalization of infectious HIV-1 particles is unknown. We demonstrate that antibodies with different paratopes, isotypes and subclasses mediate HIV-1 virion internalization, using novel HIV-1 internalization assays. Env IgG3 mediated greater virion internalization activity than IgG1, followed by IgA1 and IgA2. Given that Env IgG3 correlated with decreased risk of HIV-1 infection in the one partially efficacious HIV-1 vaccine trial to date (RV144), determining the underlying antiviral mechanisms is critical for improving HIV-1 prevention strategies. Our study provides direct evidence of a new antiviral mechanism against HIV-1 infection, IgG3 mediated virion internalization, and raises the hypothesis that a mechanism of protection mediated by IgG3 could be this improved Fc-mediated antiviral function. These findings have important implications for harnessing antibody effector functions for HIV-1 vaccine design, HIV-1 cure and passive immunotherapy for HIV-1 clearance at the portal of entry.

Following envelope mediated fusion, the HIV-1 core is released into the cytoplasm of the target cell and undergoes a series of trafficking and replicative steps that result in the nuclear import of the viral genome, which ultimately leads to the integration of the proviral DNA into the host cell genome. Previous studies have found that disruption of microtubules, or depletion of dynein or kinesin motors, perturb the normal uncoating and trafficking of the viral genome. Here, we show that the Kinesin-1 motor, KIF5B, induces a relocalization of the nuclear pore component Nup358 into the cytoplasm during HIV-1 infection. This relocalization of NUP358 is dependent on HIV-1 capsid, and NUP358 directly associates with viral cores following cytoplasmic translocation. This interaction between NUP358 and the HIV-1 core is dependent on multiple capsid binding surfaces, as this association is not observed following infection with capsid mutants in which a conserved hydrophobic binding pocket (N74D) or the cyclophilin A binding loop (P90A) is disrupted. KIF5B knockdown also prevents the nuclear entry and infection by HIV-1, but does not exert a similar effect on the N74D or P90A capsid mutants which do not rely on Nup358 for nuclear import. Finally, we observe that the relocalization of Nup358 in response to CA is dependent on cleavage protein and polyadenylation factor 6 (CPSF6), but independent of cyclophilin A. Collectively, these observations identify a previously unappreciated role for KIF5B in mediating the Nup358 dependent nuclear import of the viral genome during infection., Author Summary Fusion of viral and target cell membranes releases the HIV-1 viral capsid, which houses the viral RNA and proteins necessary for viral reverse transcription and integration, into the cytoplasm of target cells. To complete infection, the viral capsid must ultimately traffic to the nucleus and undergo a process known as uncoating to allow the nuclear import of the viral genome into the nucleus, where it subsequently integrates into the genome of the target cell. Here, we show that the concerted actions of microtubule motor KIF5B and the nuclear pore component Nup358 cooperatively facilitate the uncoating and nuclear import of the viral genome. Moreover, we also identify the determinants in the viral capsid protein, which forms the viral capsid core, that are required for KIF5B dependent nuclear entry. These studies reveal a novel role for the microtubule motor KIF5B in the nuclear import of the viral genome and reveal potential intervention targets for therapeutic intervention.

HIV and other lentiviruses can productively infect nondividing cells, whereas most other retroviruses, such as murine leukemia virus, require cell division for efficient infection. However, the determinants for this phenotype have been controversial. Here, we show that HIV-1 capsid (CA) is involved in facilitating HIV infection of nondividing cells because amino acid changes on CA severely disrupt the cell-cycle independence of HIV. One mutant in the N-terminal domain of CA in particular has lost the cell-cycle independence in all cells tested, including primary macrophages. The defect in this mutant appears to be at a stage past nuclear entry. We also find that the loss of cell-cycle independence can be cell-type specific, which suggests that a cellular factor affects the ability of HIV to infect nondividing cells. Our data suggest that CA is directly involved at some step in the viral life cycle that is important for infection of nondividing cells., Author Summary HIV and related viruses are unusual among retroviruses in their ability to replicate independently of cell-cycle progression of target cells. However, the determinants of this phenotype have been controversial. Here, we identified mutations on the surface of the capsid (CA) protein that reduce the ability of HIV to infect nondividing cells. These mutations also confer cell-cycle dependency on HIV, even in dividing cells. Interestingly, some CA mutants lose cell-cycle independence only in certain cell types. Thus, these findings suggest that a cellular factor targeting CA regulates HIV-1 infection in nondividing cells. Surprisingly, these mutations do not appear to affect nuclear localization of viral genomes, which points to a novel regulation of the cell-cycle independence of HIV by the CA protein.

To gain insight into female-to-male HIV sexual transmission and how male circumcision protects against this mode of transmission, we visualized HIV-1 interactions with foreskin and penile tissues in ex vivo tissue culture and in vivo rhesus macaque models utilizing epifluorescent microscopy. 12 foreskin and 14 cadaveric penile specimens were cultured with R5-tropic photoactivatable (PA)-GFP HIV-1 for 4 or 24 hours. Tissue cryosections were immunofluorescently imaged for epithelial and immune cell markers. Images were analyzed for total virions, proportion of penetrators, depth of virion penetration, as well as immune cell counts and depths in the tissue. We visualized individual PA virions breaching penile epithelial surfaces in the explant and macaque model. Using kernel density estimated probabilities of localizing a virion or immune cell at certain tissue depths revealed that interactions between virions and cells were more likely to occur in the inner foreskin or glans penis (from local or cadaveric donors, respectively). Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors. At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/− 0.0154 and 0.0171 +/− 0.0038 virions/image, p = 0.001). In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/− 0.0079 virions/image) than glans tissue (0.0167 +/− 0.0033 virions/image, p, Author Summary Although several clinical trials have demonstrated that male circumcision can protect men from becoming infected with HIV, we know very little about how men get infected through sex and how circumcision changes this. In this study, we explored possible sites of virus transmission across the penis by looking at how HIV interacts with adult male foreskins, penile tissues from circumcised and uncircumcised cadavers, and male rhesus macaques. Using epifluorescent microscopy, we captured images of individual HIV particles entering the penile skin, sometimes to depths where CD4+ (potential target) cells could be found. We found more virus in and on the inner aspect of the foreskin than the outer aspect of the foreskin after culturing for 24 hours. Additionally, there was more virus entering the glans penis as compared to foreskin tissues from uncircumcised cadaveric donors, and to greater depths in these tissues. We made similar observations of virus entering the tissue in living rhesus macaques, strengthening the results obtained from human tissues. This information should help us better understand how the virus moves into uncircumcised penile tissue placing uncircumcised men at higher risk for HIV infection during sex.

The majority of new HIV infections occur in women as a result of heterosexual intercourse, overcoming multiple innate barriers to infection within the mucosa. However, the avenues through which infection is established, and the nature of bottlenecks to transmission, have been the source of considerable investigation and contention. Using a high dose of a single round non-replicating SIV-based vector containing a novel dual reporter system, we determined the sites of infection by the inoculum using the rhesus macaque vaginal transmission model. Here we show that the entire female reproductive tract (FRT), including the vagina, ecto- and endocervix, along with ovaries and local draining lymph nodes can contain transduced cells only 48 hours after inoculation. The distribution of infection shows that virions quickly disseminate after exposure and can access target cells throughout the FRT, with an apparent preference for infection in squamous vaginal and ectocervical mucosa. JRFL enveloped virions infect diverse CD4 expressing cell types, with T cells resident throughout the FRT representing the primary target. These findings establish a new perspective that the entire FRT is susceptible and virus can reach as far as the ovary and local draining lymph nodes. Based on these findings, it is essential that protective mechanisms for prevention of HIV acquisition must be present at protective levels throughout the entire FRT to provide complete protection., Author Summary There is currently a great effort world-wide to develop interventions such as vaccines and microbicides to decrease, or hopefully block, HIV transmission. To model the infection of women, the field utilizes the rhesus macaque vaginal transmission model. Understanding the initial events leading to infection after viral challenge of the female reproductive tract (FRT) is crucial for the development of functional prevention strategies. To this end, we developed a novel method for detecting infection in the rhesus macaque FRT after vaginal inoculation. This method utilizes single round replication defective vector that expresses dual reporter proteins, Luciferase and mCherry. Monitoring Luciferase expression allows us to identify the sites of infection within the intact FRT, while fluorescent protein mCherry allows us to visualize the single infected cells. Our studies revealed that virus can access the entire upper and lower reproductive tract. Infection occurs primarily in vaginal and ectocervical tissue, but can spread as far as the ovary and local draining lymph nodes. All classically defined susceptible cell types can be infected with the broadly tropic HIV envelope utilized in this study. Prevention strategies aimed at protecting from HIV infection should consider the entire FRT architecture as potentially susceptible and design interventions accordingly.