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Early short-term treatment with neutralizing human
monoclonal antibodies halts SHIV infection in infant macaques
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These experiments show that NmAbs delivered subcutaneously are swiftly distributed to blood and tissues and that they maintain neutralizing activity at distal sites. They further indicate that NmAbs are effective at clearing viral foci in blood and tissues during the earliest stages of HIV penetration of the tissues, a different mechanism from that of ART.
Viral RNA has been detected in macaques as early as 1 d following vaginal exposure to SIV45, but there is a common view that HIV and SIV may have a short 'eclipse phase' of limited, localized viral replication that lasts a number of days, in which the spread of the founder infection is dependent upon target cell availability to spread to lymphatic tissues45. Our data show that, at least in this model system of oral SHIV exposure in infant macaques, virus replication is detected in lymphatic tissues at 24 h after infection and is not locally restricted. Here we found evidence of an immediate impact of a single dose of passively transferred NmAbs on seeding of the virus, with a significant difference in early tissue-associated viral RNA and DNA in treated versus nontreated infants. Early post-exposure, short-term administration of powerful NmAbs [neutralizing monoclonal antibodies] effectively cleared the virus in vivo by 14 d and prevented viral rebound after decay of the passive NmAbs. We present three lines of evidence that the ten macaques studied for >24 weeks were clear of virus. Firstly, all of the macaques failed to develop adaptive immune responses. Secondly, we showed that >300 coded tissue samples from these macaques were virus negative, using an ultrasensitive PCR methodology based on detection of the SIV gag gene. Thirdly, we depleted the CD8+ T cells in the four lower-dose macaques (group 6) and observed no viral rebound.
In fact, safety trials in HIV-exposed newborns for treatment with the NmAb VRC01 have begun in the USA and South Africa (http://impaactnetwork.org/studies/P1112.asp), following a safety trial in adults
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Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in infant macaques
Nature Medicine
21 March 2016
Ann J Hessell1,2, J Pablo Jaworski1, Erin Epson1, Kenta Matsuda3, Shilpi Pandey1, Christoph Kahl1, Jason Reed2, William F Sutton1, Katherine B Hammond2, Tracy A Cheever1, Philip T Barnette1, Alfred W Legasse1, Shannon Planer1, Jeffrey J Stanton1, Amarendra Pegu4, Xuejun Chen4, Keyun Wang4, Don Siess1, David Burke1, Byung S Park1, Michael K Axthelm1,2, Anne Lewis1, Vanessa M Hirsch3, Barney S Graham4, John R Mascola4, Jonah B Sacha1,2 & Nancy L Haigwood1,2
Abstract
Prevention of mother-to-child transmission (MTCT) of HIV remains a major objective where antenatal care is not readily accessible. We tested HIV-1-specific human neutralizing monoclonal antibodies (NmAbs) as a post-exposure therapy in an infant macaque model for intrapartum MTCT. One-month-old rhesus macaques were inoculated orally with the simian-human immunodeficiency virus SHIVSF162P3. On days 1, 4, 7 and 10 after virus exposure, we injected animals subcutaneously with NmAbs and quantified systemic distribution of NmAbs in multiple tissues within 24 h after antibody administration. Replicating virus was found in multiple tissues by day 1 in animals that were not treated. All NmAb-treated macaques were free of virus in blood and tissues at 6 months after exposure. We detected no anti-SHIV T cell responses in blood or tissues at necropsy, and no virus emerged after CD8+ T cell depletion. These results suggest that early passive immunotherapy can eliminate early viral foci and thereby prevent the establishment of viral reservoirs.
Introduction
Recent advances in the discovery of human HIV NmAbs that have high potency and breadth of coverage have rekindled an interest in their use as pre-exposure prophylaxis, as well as therapeutic agents, including in the setting of MTCT, in which the time of exposure is known1, 2. A combination of measures—including antiretroviral treatment (ART) of the mother and the infant, cesarean section and formula feeding—have diminished the rate of MTCT from 35% to less than 3% (ref. 3). Despite this reduction, HIV infects approximately 200,000 children yearly, primarily in places where ART is not available4. Treatment of babies with ART during both the early peripartum and the breast-feeding timeframes is recommended5, but risks remain, including the toxicities associated with long-term use and the development of drug-resistant viral variants6. Therefore, discovering less toxic methods to limit transmission to newborns would be advantageous2.
In mucosal HIV and SIV transmission, the virus establishes a small founder population of infected cells after it has traversed the vaginal mucosal barrier7. This localized infection rapidly expands and spreads to local draining lymph nodes (LN), before disseminating systemically by 1 week after exposure8, 9. Similarly in nonhuman primate (NHP) models of oral SIV exposure, the oral and esophageal mucosa and the tonsils are sites of early viral infection within 1 d post-exposure (d.p.i.), with rapid systemic dissemination, via the regional lymphatics, occurring within 1 week after exposure10, 11. Because IgG from the circulation contributes substantially to the immunoglobulin pool in tissue and genital tract secretions, passively transferred neutralizing antibodies (NAbs) may have a protective effect by interaction with the virus at the mucosal level12, thus preventing systemic spread. In adult NHP models of mucosal SHIV transmission, there is abundant evidence for protective prophylactic efficacy with passively transferred human NmAbs13, 14, 15, 16, 17, 18. In vitro, NmAbs have been shown to block HIV infection of dendritic cells and subsequent transmission to T cells19. Direct vaginal application of NAbs before challenge is protective in macaques20, and in HIV-exposed but uninfected humans, mucosal IgA can block transcytosis in vitro12. Vaccine-induced protection from vaginal challenge correlates with levels of glycoprotein 41 (gp41)-specific cervicovaginal IgA and IgG that have antiviral and transcytosis-blocking activities21. However, the tissue localization and the kinetics of passively transferred antibodies are still not well defined13, 22.
There is evidence for an impact by NmAbs in lowering plasma virus levels in established infections in NHP models23, 24, 25 and in humans25, 26. In NHP models, post-exposure prophylaxis using cocktails of the first-generation human NmAbs b12, 2G12, 2F5 and 4E10 partially prevented oral SHIV infection in newborns24. A single dose combining the newer, more-potent NmAbs VRC07-523 and PGT121 delivered 10 d after intravenous SHIV infection suppressed acute viremia and limited seeding of viral reservoirs in adult macaques27. We have shown that neutralizing polyclonal IgG purified from SIV- or SHIV-infected macaques that are injected subcutaneously (s.c.) can effectively control viremia and accelerate B cell responses, resulting in reduced pathogenesis in SIV-infected adults28 and in SHIV-infected newborn macaques29, 30. We hypothesized that a cocktail of two potent and broadly cross-reactive NmAbs, VRC07-523 and PGT121, would slow the initial virus expansion and reduce the chance of rapid escape in infant macaques exposed to pathogenic SHIV. We show that combined doses as low as 10 mg per kg body weight (mg/kg) administered 24 h after exposure can intercept replicating viral foci established by day 1 and prevent orally administered virus from establishing permanent viral reservoirs.
Discussion
Pre-exposure prophylaxis (PrEP) with ART is effective in limiting transmission in the setting of MTCT, as well as in healthy adults38. One of the major goals in treating HIV-1 infection is to discover methods that can clear the established viral reservoir39. To date, only a single case of a 'functional cure' has been documented following bone marrow transplantation40. Vaccine-induced, persistent T cell responses cannot prevent infection but can reduce established SIV reservoirs to undetectable levels in about half of vaccinated macaques41. NHP studies with ART suggest that treatment as early as 3 d.p.i. is too late to prevent the establishment of the reservoir, as the virus rebounded after cessation of drug treatment42. These data are consistent with the case of the 'Mississippi baby', in which ART therapy was started within 30 h of birth but did not prevent HIV infection43, 44. Thus, the time for intervention is extremely limited, and ART alone may not be effective at eliminating a founder infection.
Viral RNA has been detected in macaques as early as 1 d following vaginal exposure to SIV45, but there is a common view that HIV and SIV may have a short 'eclipse phase' of limited, localized viral replication that lasts a number of days, in which the spread of the founder infection is dependent upon target cell availability to spread to lymphatic tissues45. Our data show that, at least in this model system of oral SHIV exposure in infant macaques, virus replication is detected in lymphatic tissues at 24 h after infection and is not locally restricted. Here we found evidence of an immediate impact of a single dose of passively transferred NmAbs on seeding of the virus, with a significant difference in early tissue-associated viral RNA and DNA in treated versus nontreated infants. Early post-exposure, short-term administration of powerful NmAbs effectively cleared the virus in vivo by 14 d and prevented viral rebound after decay of the passive NmAbs. We present three lines of evidence that the ten macaques studied for >24 weeks were clear of virus. Firstly, all of the macaques failed to develop adaptive immune responses. Secondly, we showed that >300 coded tissue samples from these macaques were virus negative, using an ultrasensitive PCR methodology based on detection of the SIV gag gene. Thirdly, we depleted the CD8+ T cells in the four lower-dose macaques (group 6) and observed no viral rebound.
These experiments show that NmAbs delivered subcutaneously are swiftly distributed to blood and tissues and that they maintain neutralizing activity at distal sites. They further indicate that NmAbs are effective at clearing viral foci in blood and tissues during the earliest stages of HIV penetration of the tissues, a different mechanism from that of ART. We hypothesize that antibody-mediated effector functions, including cytotoxicity and phagocytosis, are required for killing infected cells that express HIV envelope gp160 on their surface46. If so, then NmAbs present for an extended period of time after exposure would have the capability to destroy infected cells and neutralize virus particles emanating from cells in which infection was established within the first few hours following an exposure event. In this setting, post-exposure therapy, using a NmAb cocktail administered 24 h after SHIV exposure and continuing for 2 weeks, resulted in the maintenance of high NmAb concentrations in vivo for at least 2 weeks. The importance of repeated dosing is not known, but in the case of breast-feeding mothers, there is continued opportunity for transmission of HIV-1. It will be important to understand whether repeated NmAb dosing in babies could expand the protective window.
Several relevant questions remain unanswered for the treatment of HIV-infected newborns and children born to HIV-positive mothers, including the practical and cultural issues of treating breast-feeding mothers and babies, as well as a determination of optimal antibody cocktail formulations. Any future use of human NmAbs in the clinic will presumably require several antibodies, or engineered antibodies with multiple specificities, to avoid the potential for the emergence of viral escape mutants. Because ART has a short half-life and requires strict adherence to the drug regimen to be effective, supplementation with passive NmAbs with relatively long half-lives may widen the therapeutic window.
Identifying human NmAbs that can impact infection in a macaque model for MTCT can provide a proof of principle for the value of using antibodies to augment ART. In fact, safety trials in HIV-exposed newborns for treatment with the NmAb VRC01 have begun in the USA and South Africa (http://impaactnetwork.org/studies/P1112.asp), following a safety trial in adults47. Our findings begin to define the window of opportunity for effective treatment after intrapartum exposure. If these results can be applied to clinical settings, then there is optimism that early passive immunotherapy may provide protection from HIV infection, even in the absence of ART.
Results
Titration and biodistribution of subcutaneously administered antibodies in macaques
We initially conducted studies to define the protective dose and kinetics of the CD4-binding site-directed NmAb VRC01 in blocking newborn macaques from oral SHIVSF162P3 infection after s.c injection and to determine the kinetics of passively transferred IgG in naive and infected macaques. First, we administered VRC01 to a total of seven male and female one-month-old macaques at 20 mg/kg (n = 2) or 5 mg/kg (n = 5) 24 h before SHIV exposure. We measured SHIVSF162P3 envelope-specific binding and neutralizing antibody kinetics in vivo. The time to maximal concentration in the plasma was 24 h, independent of dose, and the serum (plasma) half-life of VRC01 was 3.9-4.2 d (Supplementary Fig. 1). Neither of the two macaques injected with the 20 mg/kg dose and only one of five macaques injected with the 5 mg/kg dose became infected. In this macaque, the magnitude and kinetics of virus in the plasma, termed the plasma virus load (PVL), were indistinguishable from that of control animals treated with IgG purified from naive macaques30 (Supplementary Fig. 1). These data are consistent with results from passive protection studies using VRC01 in juvenile and adult macaques18 and guided the therapeutic range we used for infant macaques.
Next, in a separate study designed to determine whether the kinetics of passively transferred IgG is altered in the presence of viral antigen, we assessed the distribution of purified polyclonal Ig from SIV-infected macaques (referred to as SIVIG) in a total of four male and female macaques, and we compared SIVIG kinetics in SIV-infected macaques to that in naive macaques. SIVIG was rapidly distributed in the plasma and tissues of infected and naive animals (Supplementary Fig. 2). We used in situ hybridization to localize SIV in tissue samples collected at 24 h and at 2 weeks after oral challenge with SIVsmE660. SIV was undetectable in 24-h tissue samples but was detectable after 2 weeks in tissues both adjacent to and distant from the site of challenge (Supplementary Fig. 3). Thus, IgG delivered subcutaneously is rapidly and widely distributed, and is unimpeded by viral antigen.
NmAb cocktail immunotherapy in the presence of SHIV
We next assessed the effectiveness of HIV-1 NmAbs as post-exposure prophylaxis in one-month-old infant inoculated orally with SHIVSF162P3. For in vivotherapy, we tested a cocktail of VRC07-523 and PGT121, two potent NmAbs that target different regions of the HIV-1 envelope and that have been shown to have additive effects in vitro31. VRC07-523 is an engineered clonal relative of VRC01 that shows increased neutralization of most HIV strains and improved in vivo protection capabilities32. Therefore we used VRC07 instead of VRC01 for these therapeutic studies. PGT121 interacts with the variable regions and glycans of HIV-1 gp120 (refs. 33,34) and protects adult macaques from mucosal challenge at very low plasma titers35. Cocktails of PGT121 and VRC07-523 were prepared at total doses of 10 mg/kg (5 mg/kg each antibody) and 40 mg/kg (20 mg/kg each antibody) and delivered subcutaneously.
We inoculated 20 one-month-old rhesus macaques orally with SHIVSF162P3 on day 0 and followed them for up to 28 weeks to assess virological, immunological and disease outcomes, with or without NmAb treatment starting on day 1. Pairs of animals were killed at days 1, 2 or 14 after exposure to monitor the development of SHIVSF162P3 infection in the blood and tissues of treated and untreated macaques (Table 1; groups 1-4). We delivered NmAbs on days 1, 4, 7 and 10 after SHIV exposure (Fig. 1a and Table 1; groups 4-6). SHIVSF162P3 infection by the oral route in one-month-old macaques results in reproducible, sustained PVL at >107 copies/ml plasma and ~104 copies per μg DNA for at least 24 weeks in all of the animals30. To conserve animals, historical controls were used as a comparison group for the 24-week follow-up (Table 1; group 7).
We evaluated the kinetics of the individual NmAbs and the cocktail in plasma from all 12 treated infants that were on the study for at least 2 weeks (Table 1; groups 4, 5 and 6). Peak NmAb cocktail concentrations in plasma occurred by 24 h after the s.c. injection in all animals at both NmAb doses. In the four macaques that received the 10 mg/kg dose, the average cocktail concentration during the first 2 weeks was 44 μg/ml, and in the eight macaques that received the 40 mg/kg dose, it was 113 μg/ml (Fig. 1b). By using reagents designed to specifically detect each NmAb independently, we found that PGT121 concentrations in the plasma were consistently higher at both doses than those of VRC07-523. Multiple dosing prevented us from calculating the in vivo half-lives of each NmAb, but PGT121 was detectable in plasma for 2 weeks longer than VRC07-523 in several macaques. PGT121, administered at 5 mg/kg, was maintained for >20 weeks in the plasma from a single macaque, 33537 (Fig. 1b, bottom left). An unusually slow antibody decay rate in the plasma of subjects that were passively infused with PGT121 has been recently reported, in which plasma concentrations of 5-20 μg/ml were still present after 10 weeks27.
We assessed SHIVSF162P3-neutralization activity in the plasma of all infant macaques and found that it decayed by 6-7 weeks in all of the animals except macaque 33537, in which declining neutralization of SHIVSF162P3 was detected at titers of 102-103 before becoming undetectable at week 20 (Supplementary Fig. 4). Calculation of the average 50% inhibitory concentration in plasma (IC50) of the NmAb cocktail during the first 2 weeks after SHIVSF162P3 exposure was 0.0134 and 0.0120 μg/ml in the 10 mg/kg and 40 mg/kg groups, respectively, which is close to the IC50 (0.0128 μg/ml) obtained from purified NmAbs specific for SHIVSF162P3 in the TZM-bl standardized cell line that expresses luciferase in the presence of HIV or SIV Tat and that is used to quantify NAbs in vitro (Supplementary Fig. 4).
To measure transudation of NmAb into tissues and to assess neutralization potency within tissues and organs during the first 2 weeks, we extracted specimens from six macaques at different necropsy time points. Analysis of the antibody extracted from two macaques (group 4) that were sacrificed at 14 d after four doses of NmAbs showed that NmAbs were systemically distributed at concentrations from 48-700 ng/ml of tissue lysate (Supplementary Table 1). Two macaques (group 2a) exposed to SHIVSF162P3, treated once with a NmAb cocktail dose of 10 mg/kg 1 d later and sacrificed on day 2 had NmAbs in tissue lysates in concentrations up to 791 ng/ml. Two macaques (group 2b) treated once with 10 mg/kg, without SHIV exposure, had NmAbs in tissues at levels similar to those in macaques 34263 and 34290, which were pre-exposed to SHIV. We assessed the neutralizing activity to SHIVSF162P3 in tissue homogenates from ~100 mg of necropsy samples from the animals sacrificed at 1, 2 and 14 d after exposure (groups 2 and 4). The 50% neutralization titers (ID50) for SHIVSF162P3 of tissue lysates averaged ~1:50 in the tested samples at 1 d after s.c. NmAb injections, increased to an average of ~1:100 by day 2 and were 1:150 at day 14, with good agreement between the titers observed for macaques that were sacrificed at the same time points (Table 2). However, NmAb titers in the colon and reproductive tract from macaque 34290 were about 3-5 times higher than those from macaque 34263, which was necropsied at the same time point. Tissue-associated IC50 concentrations of the NmAb cocktail in the samples tested ranged from 0.5-10.0 ng/ml, which is similar to the IC50 of the NmAb cocktail (Supplementary Fig. 4). We conclude that the presence of SHIV during the first day after oral exposure did not affect NmAb distribution or levels in vivo and that the NmAb cocktail was rapidly distributed to tissues.
We collected multiple tissues from all of the macaques at necropsy (Fig. 2c), and in samples collected within 2 d of SHIVSF162P3 exposure, we measured low levels of SHIVSF162P3 DNA in mucosa and LN that were proximal and distal to the oral exposure site (Fig. 2d,e) in treated and untreated animals. In comparison to treated animals, the virus was widespread at day 14 in untreated animals (Fig. 2f) and peaked at >3,000 copies/μg of DNA throughout the LN and gut, consistent with levels of DNA in the tissues of adult and six-month-old Macaca nemestrina with high levels of plasma viremia36. As seen in the blood, following NmAb treatment on days 1, 4, 7 and 10 at 40 mg/kg, virus was not detectable in any tissue at day 14 (Fig. 2g and Supplementary Table 2). To determine whether the viral DNA-positive tissues contained replicating SHIV, we measured viral RNA in several tissue samples taken from these same macaques that were sacrificed on 1, 2 or 14 d after virus exposure. Viral DNA and RNA levels, tested as blinded samples from these tissues, show that productive SHIVSF162P3 infection had begun in multiple tissues by day 1, increasing exponentially by day 14 (Table 2). However, in two macaques that were treated with 10 mg/kg on day 1 (group 2a; sacrificed on day 2), there was no viral RNA detected in the samples tested. Notably, the NmAbs were colocalized in these virus-positive tissues, suggesting the potential for antibody effects as early as 1 d after treatment. Moreover, the results suggest that virus present early after exposure can be intercepted and cleared by NmAbs present in the same tissues (Table 2).
Prevention of productive infection, viral rebound and pathogenesis
To evaluate the effect of early short-term NmAb therapy on viral control, we monitored SHIVSF162P3 in blood, LN and tissues in animals followed for 24-28 weeks. PVL in the controls routinely peaked at 2 weeks post-infection (w.p.i.) and persisted at levels that ranged from 106-108 copies/ml. In newborns that were treated with 10 mg/kg (Fig. 3a) or 40 mg/kg (Fig. 3b) NmAbs, there was no plasma viremia detected in any of the samples collected over the course of the study. A single time point in the 40 mg/kg group was positive for only one of two replicates, and additional material to retest this sample was not available. Longitudinal cell-associated viral loads (CAVL) in PBMC DNA were negative for each of the >300 samples tested from the ten macaques in groups 5 and 6 (Fig. 3c,d). In short, all of the NmAb-treated infants had undetectable PVL or CAVL in blood.
We also measured the levels of SHIVSF162P3 DNA in >300 homogenized tissue samples obtained at 24-28 w.p.i. and in inguinal LNs collected 12 w.p.i. from all ten macaques, using ultrasensitive qPCR37. Tissue samples from all SHIV-exposed infants that received the 2-week course of NmAb cocktail were tested as coded samples and were negative for virus in both dosage groups (Supplementary Table 2). As discussed above, only very low levels of virus were detected in tissue specimens from the group 2a macaques 34263 and 34290, which were sacrificed at 2 d.p.i. after a single NmAb dose (Fig. 2e). Similarly to that seen in blood, virus was widespread in tissues at 14 d.p.i. in group 3 control macaques that did not receive NmAbs (Fig. 2f). As early as 1 d.p.i., tissue-associated virus in mucosal tissue adjacent to the exposure site, the draining LN and the gut tissue was evident in macaques from group 1 (untreated controls) (Fig. 2d and Supplementary Table 2). As compared to the untreated (group 1) macaques killed at 1 d.p.i., NmAb-treated (group 2a) macaques that were sacrificed 2 d.p.i had significantly lower amounts of tissue-associated virus (P = 0.0061; Fig. 4). The discovery of traces of virus at 2 d.p.i (Fig. 2e) and none at 14 d.p.i. (Fig. 2g) implies that NmAbs intercepted and neutralized SHIVSF162P3 replication, cleared infected cells and halted the spread of infection in these macaques and in all ten NmAb-treated infants that were followed for 6 months. Consistent with these data, pathology results also showed the absence of organ or tissue pathology (Supplementary Fig. 5).
No evidence for T cell immunity or viral rebound
To evaluate T cell immunity in the SHIV-exposed macaques, we used intracellular cytokine staining (ICS) to measure PBMC, spleen and mesenteric LN responses specific for the SIVmac239 proteins Gag and Vif (present in the SHIV chimera) in the ten macaques that were studied for 6 months. No SHIV-specific T cell responses were detected in PBMC at week 20 or in tissue samples after necropsy (Supplementary Fig. 6). To determine whether there were any reservoirs controlled by CD8+ T cell-mediated suppression, we depleted CD8+ cells to undetectable levels in the four animals in the 10 mg/kg group and monitored them for viremia for 4 weeks (Supplementary Fig. 6). There was no evidence of virus rebound in the plasma of these animals during the CD8+ depletion phase (Fig. 3a,c), further supporting the concept that early passive NmAb therapy with the cocktail of VRC07-523 and PGT121 disrupted establishment of virus reservoirs, thereby preventing exposure to antigen and development of cellular immunity.
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