JVI Accepted Manuscript Posted Online 18 November 2015 J. Virol. doi:10.1128/JVI.02454-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Title: HIV-1 monoclonal antibodies suppress acute SHIV viremia and limit seeding of cell-

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associated viral reservoirs

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Byline: Diane L. Bolton,1†# Amarendra Pegu,2† Keyun Wang,2 Kathleen McGinnis,2 Martha

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Nason,3 Kathryn Foulds,2 Valerie Letukas,2 Stephen D. Schmidt,2 Xuejun Chen,2 John Paul

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Todd,2 Jeffrey Lifson,4 Srinivas Rao,2§ Nelson L. Michael,5 Merlin L. Robb,1 John R. Mascola,2

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and Richard A. Koup2

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US Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring,

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Maryland 20910. Henry M. Jackson Foundation for the Advancement of Military Medicine,

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Bethesda, Maryland 20818.

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Institutes of Health, Bethesda, Maryland 20892.

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Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.

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Laboratory for Cancer Research, AIDS and Cancer Virus Program, P.O. Box B, Frederick,

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Maryland 21702.

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5

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Maryland 20910.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National

Biostatistics Research Branch, Division of Clinical Research, National Institute of Allergy and

AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc./Frederick National

US Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring,

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Running title: Acute SHIV passive immunotherapy with HIV-1 Env mAbs

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#Address correspondence to Diane L. Bolton, [email protected]

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†D.L.B.

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§

and A.P. contributed equally to this work.

Present address: SanofiPasteur, 38 Sidney St #370, Cambridge, MA 02139.

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Abstract: Combination antiretroviral therapy (cART) administered shortly after HIV-1

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infection can suppress viremia and limit seeding of the viral reservoir, but lifelong treatment is

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required in the majority of patients. Highly potent broadly neutralizing HIV-1 monoclonal

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antibodies (mAbs) can reduce plasma viremia when administered during chronic HIV-1

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infection, but the therapeutic potential of these antibodies during acute infection is unknown.

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We tested the ability of HIV-1 envelope glycoprotein specific broadly neutralizing mAbs to

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suppress acute simian-human immunodeficiency virus (SHIV) replication in rhesus macaques.

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Four groups of macaques were infected with SHIV-SF162P3 and received i) the CD4-binding

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site mAb VRC01 or ii) a combination of a more potent clonal relative of VRC01 (VRC07-523)

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and a V3 glycan-dependent mAb (PGT121) or iii) daily cART, all on day 10, just prior to

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expected peak plasma viremia, or iv) no treatment. Daily cART was initiated 11 days after mAb

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administration and was continued for 13 weeks in all treated animals. Over a period of 11 days

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after a single administration, mAb treatment significantly reduced peak viremia, accelerated the

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decay slope and reduced total viral replication as compared to untreated controls. Proviral DNA

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in lymph node CD4 T cells was also diminished after treatment with dual mAb. These data

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demonstrate the virological effect of potent mAbs and support future clinical trials that

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investigate HIV-1 neutralizing mAbs as adjunctive therapy with cART during acute HIV-1

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infection.

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Importance: Treatment of chronic HIV-1 infection with potent broadly neutralizing HIV-1

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mAbs has been shown to significantly reduce plasma viremia. However, the anti-viral effect of

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mAb treatment during acute HIV-1 infection is unknown. Here, we demonstrate that mAbs

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targeting the HIV-1 envelope glycoprotein both suppress acute SHIV plasma viremia and limit

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CD4 T cell-associated viral DNA. These findings provide support for clinical trials of mAbs as

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adjunctive therapy with antiretroviral therapy during acute HIV-1 infection.

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Introduction Virological events in the first weeks following HIV-1 transmission set the stage for

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lifelong chronic infection that remains incurable with currently available combination

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antiretroviral therapy (cART), at least in part due to early establishment of viral reservoirs,

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including latently infected cells, that persist despite cART and can give rise to recrudescent

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infection when treatment is interrupted. A major hurdle to eradicating infection is the early

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establishment of persistent viral reservoirs. Clinical cohorts in whom HIV-1 infection is

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diagnosed during this early acute phase provide a unique opportunity to modify the course of

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disease and better understand how viral reservoirs are established. While initiation of cART

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during Fiebig I-III limits residual cell-associated viral DNA levels (1, 2), current antiretrovirals

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act by blocking new rounds of infection and are thus limited in their ability to affect populations

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of already infected cells that can contribute to viral reservoirs. Moreover, recent evidence from

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experimentally infected rhesus macaques indicates that seeding of viral reservoirs can occur

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before the advent of detectable viremia (3). While efforts to induce virus expression from

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latently infected cells to facilitate elimination by immune-mediated cytotoxic or viral cytopathic

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mechanisms are being actively explored, their efficacy remains to be demonstrated (4-7).

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Alternative treatment options are needed, with particular focus on agents capable of targeting

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already infected cells.

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The recent isolation from chronically infected patients of potent broadly neutralizing

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monoclonal antibodies (mAbs) specific for the HIV-1 Envelope glycoproteins creates new

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possibilities for therapeutic agents (8-11). In addition to direct neutralization of virus, antibodies

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can facilitate the recognition and elimination of infected cells through Fc-mediated mechanisms

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such as antibody-dependent cell-mediated cytotoxicity and complement-mediated lysis (12-14).

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In rhesus macaque and humanized mice animal models of SHIV and HIV-1 infection

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respectively, HIV-1 mAbs have been shown to be effective as both immunoprophylactic and

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immunotherapeutic agents (15-20). It has also been suggested that the number of infected cells

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is reduced by mAb therapy in chronically infected animals (15, 17). These studies have led to

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initiation of clinical trials testing safety and efficacy of mAbs for both prevention and treatment

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of HIV-1 in humans (21-27). A recent report on the use of 3BNC117 to suppress viremia in

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chronically HIV-1 infected individuals validates the animal data and provides rationale for use of

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mAb therapy against HIV-1 infection (28). However, the impact of mAbs administered during

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early acute infection on virus replication, reservoir establishment, and adaptive immune

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responses is not known.

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Here, we assessed the therapeutic activity of a single mAb, or a combination of two more

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potent mAbs, during early acute SHIV162P3 infection in rhesus macaques. The single CD4

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binding site directed mAb VRC01 was chosen because it is currently being studied in human

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clinical trials. The combination of VRC07-523 and PGT121 was chosen because both mAbs are

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under clinical development. VRC07-523 is an engineered variant related to VRC01 that is 5-10

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fold more potent (29, 30). PGT121 is a potent mAb directed to the V3-glycan supersite region of

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Env (31). We found that acute viremia was significantly reduced by a single infusion of either

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VRC01 or a combination of VRC07-523 and PGT121, and that the dual mAb therapy regimen

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dramatically diminished viral replication similarly to daily three-drug cART.

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Materials and Methods

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SHIV infection and treatment

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Indian origin rhesus macaques were randomly assigned to each treatment group and challenged

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intravenously with 20 TCID50 of SHIV-SF162P3. cART consisted of 20 mg/kg/day tenofovir

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(PMPA, given subcutaneously; from Gilead Sciences, CA), 30 mg/kg/day emtricitabine (FTC,

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given subcutaneously; from Gilead) and 100 mg raltegravir twice daily (Isentress, mixed with

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food; from Merck, NJ). Animal weights ranged from 3.35-4.49 kg. For the antibody infusions,

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low endotoxin preparation ( 0.05). To ensure that the elevated Env-

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specific antibody levels reflect adaptive rhesus responses and not detection of residual infused

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human mAb, we confirmed that the anti-rhesus Ig detection reagent used in our assay does not

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cross-react with human Ig (data not shown). Similarly, the frequency of SHIV-specific T cells was reduced several fold in treated

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animals (Fig. 3C-D). Antigen-specific CD4 and CD8 T cells were measured by in vitro peptide

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stimulation of PBMC followed by intracellular cytokine staining for IFNγ, TNFα, and IL-2.

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CD8 T cells targeting SIV Gag and HIV-1 Env were significantly diminished eight and twelve

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weeks post-infection, while a similar trend was observed for both CD4 and CD8 T cells

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following mAb treatment at week three. Treatment groups did not differ from one another. This

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suggests that mAb therapy was effective at reducing the SHIV antigen load responsible for

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stimulating adaptive T and B cell responses and that the suppression was similar to that seen with

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cART.

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The combination of cytokines expressed by a T cell has been shown to influence cellular

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long-term memory potential and renewal capacity (39-41). To determine the impact of early

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therapy on T cell functionality, we compared the cytokine distribution of Gag-specific CD4 T

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cells. Acute infection response profiles were significantly different under cART and VRC07-

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523+PGT121 compared to untreated infection (Fig. 3E, week 3). Analysis of the individual

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categories revealed a shift toward IL-2-expressing cells by these regimens, while VRC01 therapy

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and no treatment resulted in responses more likely to express only IFNγ or both IFNγ and TNFα

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(Fig. 3F). These differences are consistent with less antigen-driven differentiation of CD4 T

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cells under cART or VRC07-523+PGT121, the most potent suppressors of viremia. Following

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viremia suppression by cART in the VRC01/cART group, CD4 T cell functionality evolved to

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be more similar to the other treated groups (Fig. 3E-F, weeks 8-12), while untreated infection

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maintained a high proportion of differentiated IFNγ+TNFα+ cells. Similarly, fewer terminally

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differentiated single IFNγ+ SHIV-specific CD8 T cells were observed following VRC07-

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523+PGT121 therapy (Fig. 3E-F, week 3). Thus acute immunotherapy with either VRC07-523

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and PGT121 or daily ART facilitated the maintenance of CD4 and CD8 T cell memory potential.

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Cell-associated viral load A major objective of therapy initiation during acute HIV-1 infection is to reduce the

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establishment of a latently infected pool of cells, or reservoir. To determine the impact of mAb

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immunotherapy on the number of infected cells, we measured CD4 T cell associated viral DNA

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in lymph nodes and peripheral blood. CD4 T cells were FACS sorted into total, naïve, central

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memory, and T follicular helper (Tfh; lymph node only) populations (Fig. 4A) and viral gag

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(unspliced) DNA was measured by qPCR. The average proviral burden per cell was calculated

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by dividing the gag DNA copy number within each population by the number of cells analyzed,

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as determined by alb DNA copies. One week after treatment initiation (day 18), the normalized

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level of SIV gag DNA in lymph node and PBMC CD4 T cells was reduced by all interventions

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compared to untreated controls, to a statistically significant level for most comparisons (Fig. 4B-

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C, p0.05). Of note, VRC07-523+PGT121 treatment achieved the

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greatest day 18 reduction in lymph node naïve CD4 T cell viral load, which was less than the

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assay’s detection threshold (5E-5 copies/cell) in four of the six animals. After cART initiation in

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the mAb-treated animals, the decreased cell-associated viral DNA relative to the control group

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was maintained and remained comparable to the group that received cART only in both LN and

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PBMC (Fig. 4D). Pre-treatment (day 8) PBMC-associated viral DNA did not differ between any

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of the study groups, indicating comparable baseline proviral DNA.

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To determine whether the limited viral reservoir would impair viral resurgence upon

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cART interruption, cART was discontinued 16 weeks after infection for all treatment groups.

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Viremia rebound was observed for all animals ~12 days after cART cessation, with no difference

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between mAb and cART groups with respect to the time to peak rebound viremia or the

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magnitude of peak rebound viremia (Fig. 5).

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Discussion

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Therapeutic intervention during the acute stage of HIV-1 infection has the potential to

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limit establishment of viral reservoirs and improve prospects for success in reservoir targeting

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interventions. Our studies show robust in vivo activity of anti-HIV-1 broadly neutralizing mAbs

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during acute SHIV-SF162P3 infection. All measured virological parameters, including peak

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viremia, post-peak acute viremia, and cell-associated viral load, were reduced by dual VRC07-

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523+PGT121 antibody treatment. While VRC01 monotherapy also curtailed viral replication, it

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was less consistent than VRC07-523+PGT121, in line with the lower potency of this mAb.

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VRC01 was used alone in this study because it is planned for use in clinical trials, both as an

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early HIV-1 immunotherapeutic intervention as well as a prophylactic agent to prevent infection.

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Since SHIV SF162P3 is about 10-fold less sensitive to VRC01 than most HIV-1 isolates (30,

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38), we also used a combination of mAbs, VRC07-523 and PGT121, with more potent activity

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against SHIV-SF163P3. These two mAbs are being developed for clinical use and represent the

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potential efficacy against HIV-1 when two potent mAbs are used together. For therapeutic

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purposes, it may also be important to use antibodies to more than one site to avoid the selection

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of resistant strains in the setting of active virus replication and a diverse swarm of viral genetic

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variants.

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Recent studies using the new generation of potent HIV-1-specific monoclonal antibodies

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in humanized mice, rhesus macaques, and humans demonstrated robust, albeit typically transient,

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suppression of chronic viremia (15-17, 28). Early HIV-1 diagnosis prior to the peak of viremia

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is now feasible in a field setting and has been achieved in clinical cohorts spanning multiple

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countries (Robb et al., submitted). Here, we directly compared mAbs to cART, a benchmark for

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effective therapy, during the early phase of acute viremia, just prior to peak, and observed

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remarkably similar viremia suppression. Viremia was still declining 11 days following VRC07-

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523+PGT121 infusion, suggesting that the mAb antiviral activity was sustained for over a week.

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PGT121 alone was previously reported to completely suppress chronic SHIV viremia (15), and

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thus the relative contribution of VRC07-523 and PGT121 to the suppression observed here will

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be important to assess in future studies. Greater acute viremia control by the dual mAb arm

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relative to VRC01 is consistent with more potent in vitro neutralization of the SHIV challenge

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virus by both the VRC07-523 and PGT121 mAbs, and thus in vivo therapeutic efficacy was

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associated with neutralization activity. The ability of mAbs to inhibit acute virus replication to

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this extent indicates that they represent a potent therapy option during the exponential viral

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growth phase. To our knowledge, this is the first demonstration of mAbs suppressing acute virus

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replication. And since mAbs likely employ different antiviral mechanisms than drugs, as

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discussed below, they have the potential to augment the impact of cART when used together

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during acute infection.

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While antibody-mediated selection of resistant viral sequences is an important

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consideration for passive immunotherapy in chronic HIV-1 infection (28), we think immune

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pressure by mAbs was unlikely to give rise to less sensitive SHIV variants in this acute infection

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model due to the clonal nature of the challenge virus and the short 11-day mAb treatment

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window. Sequence analysis of the challenge stock identified a single minor Env variant, A64W,

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with no known association with resistance to the mAbs studied herein (data not shown).

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Our rhesus macaque animal model likely does not adequately recapitulate some

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important aspects of the use of human antibodies in HIV-1 infection. First, fairly rapid macaque

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immune responses generated against the human antibodies preclude prolonged antibody activity

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in vivo. Humoral responses specific for these mAbs were detected within three weeks of

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infusion in all animals. As a result, mAb half-life is more limited in macaques compared to

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humans. Future studies with simianized antibodies and variants employing mutations to increase

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FcRn binding and persistence in vivo will help address the long-term potential of mAbs to

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suppress viral replication (42). Second, some effector functions in the Fc portion of the human

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mAb may not be optimal in rhesus macaques. While both IgG and cell-surface Fcγ receptors

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show considerable sequence similarity between humans and macaques, there are species

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differences with respect to Fcγ receptor tissue expression as well as specificity and affinity for

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human IgG (43-45), which may have limited the spectrum of human mAb mediated antiviral

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activities. Third, we used intravenous inoculation to establish a model with consistent and

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relatively synchronous viremia, whereas most human infection is via a mucosal route. Peak viral

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loads typically occur slightly earlier with intravenous challenge compared to mucosal delivery.

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While cART is the cornerstone of HIV-1 treatment, antibodies may offer some potential

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benefits when added to antiretroviral drugs. Currently available antiretrovirals act only to block

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new rounds of infection, whereas mAb have the potential to inhibit new rounds of infection

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through both neutralization and other virion clearance mechanisms, as well as through the

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targeting of infected cells expressing viral antigens, via Fc receptor mediated effector functions

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(e.g. antibody-dependent phagocytosis, cell-mediated cytotoxicity, and complement-dependent

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cytotoxicity). These complementary mechanisms may enable mAbs to further impair virus

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replication when used in concert with cART. In addition, some data suggest limited ART

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penetration of lymph node (46), and thus antibodies may have greater access to infected cells

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residing in secondary lymphoid tissues than drugs. We observed a lower frequency of SHIV-

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infected naïve CD4 T cells in lymph node following VRC07-523+PGT121 compared to cART.

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However, it is not clear why only naïve cells would be targeted and not other CD4 T cell subsets.

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For these reasons, it will be important to measure mAb localization in tissues in future studies,

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including an assessment of tissue penetration as a determinant of mAb antiviral activity. We also

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saw evidence of enhanced humoral responses during acute infection with mAb therapy versus

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cART (Fig. 3), with elevated Env-specific antibodies following either VRC01 or VRC07-

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523+PGT121. We speculate that monoclonal antibody-antigen immune complexes may thus

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also aid in elicitation of the endogenous adaptive humoral immune response. Antibodies

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therefore warrant consideration for use in conjunction with cART to enhance the antiviral

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activity of drugs.

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In summary, our results show that mAb immunotherapy is effective in controlling acute

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SHIV replication. Our acute SHIV rhesus macaque model reproduced key aspects of human

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HIV-1 infection, including robust peak and set-point viremia and effective viral suppression by

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cART. Moreover, our intent to model interventions in early acute infection clinical cohorts,

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during Fiebig stages I-III, was achieved by initiating therapy during the days immediately prior

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to peak viremia. These data provide rationale for assessing the efficacy of combined cART and

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mAb therapy as well as evaluating efficacy following mucosal infection to further determine the

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utility of antibody therapy. These pre-clinical studies will inform clinical trials currently being

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planned in acutely infected individuals that will be required to establish therapeutic efficacy

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during acute infection.

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Funding information. This work was supported by a cooperative agreement (W81XWH-07-2-

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0067) between the Henry M. Jackson Foundation for the Advancement of Military Medicine,

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Inc., and the U.S. Department of Defense (DOD). This research was funded, in part, by the U.S.

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National Institute of Allergy and Infectious Diseases and in part with federal funds under

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Contract No. HHSN261200800001E from the National Cancer Institute. The funders had no

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role in study design, data collection and interpretation, or the decision to submit the work for

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publication.

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Acknowledgements. We thank Romas Geleziunas and Martha Vazquez from Gilead Sciences,

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Inc. for generously providing antiretroviral drugs for use in in vivo experiments. We thank the

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VRC Flow Cytometry Core’s Steve Perfetto and Richard Nguyen for rigorous maintenance of

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flow cytometers used for measuring antigen-specific T cell responses and sorting T cell

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populations for viral DNA quantitation. We thank Takuya Yamamoto for assistance with the

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flow cytometric staining panel for isolating T follicular helper cells. Eli Boritz and Sam Darko

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sequenced and analyzed diversity of the challenge virus stock. Plasma viral load measurements

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were performed by Mike Piatak (deceased) with technical assistance from Randy Fast, Kelli

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Oswald and Rebecca Shoemaker of the Quantitative Molecular Diagnostics Core of the AIDS

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and Cancer Virus Program of the Frederick National Laboratory for Cancer Research. We thank

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Brenda Hartman for assistance formatting manuscript figures. We thank Barney Graham for

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critical reading of the manuscript and input on study design. The views expressed are those of

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the authors and should not be construed to represent the positions of the Departments of the

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Army, Defense, or Health and Human Services.

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27

636

Table 1. Neutralization potency of anti-HIV-1 mAbs. HIV-1 viruses (>195 strains)

SHIV SF162P3 Antibody

IC50 (mg/ml)

IC80 (mg/ml)

Median IC50 (mg/ml)

Median IC80 (mg/ml)

VRC01

4.28

8.63

0.378

1.01

VRC07-523

0.520

0.975

0.045

0.219

PGT121

0.101

0.190

0.014

0.094

637 638 639 640 641

Figures:

642

levels in plasma. (A) Twenty-four rhesus macaques were challenged intravenously with

643

SHIVSF162P3 and treated on day 10 with either daily ART (n=6), a single infusion of mAb VRC01

644

(n = 6), or a single infusion of two mAbs together (VRC07-523 and PGT121). Six control

645

animals remained untreated. For those animals given mAb treatment, daily ART was initiated 21

646

days after infection. (B) Antibody levels in plasma of rhesus macaques after a single infusion of

647

mAb VRC01, or a combination of mAbs VRC07-523 and PGT121, infused 10 days post SHIV-

648

challenge. Each line represents data for one animal and error bars represent the 95% confidence

649

intervals. The shaded areas correspond to the duration of daily ART treatment. Red dotted line

650

indicates the in vitro neutralization IC80 (µg/ml) against the SHIV-SF162P3 challenge stock.

651

The black dotted line indicates the limit of detection of plasma antibody levels. (C) Geometric

652

mean antibody levels in the plasma for the mAb treatment groups (n=6 per group). The arrow

653

indicates the time of antibody injection and the shaded area represents the period of daily ART.

654

(D) Measured plasma neutralization titers against the SHIV SF162P3 challenge stock.

655

Geometric mean plasma neutralization ID80 titers are shown for the mAb treatment groups (n=6

656

per group; solid red and blue lines) at the indicated time following infection. Predicted ID80

Fig. 1. Experimental treatment schema and VRC01, VRC07-523, and PGT121 antibody

28

657

values calculated from the plasma mAb concentration shown in (B) are overlaid as dotted lines.

658

The predicted ID80 values for the group that received VRC07-523 and PGT121 was a sum of the

659

predicted ID80 value for each mAb. (E) Anti-antibody responses after SHIV challenge. The

660

geometric mean end point titer values for plasma reactivity to VRC01, VRC07-523, and PGT121

661

in infused animals was determined using an ELISA-based approach. The error bars represent the

662

95% confidence interval; the dotted line indicates the limit of detection for the assay. Arrows

663

indicate timing of mAb infusion.

664 665

Fig. 2. SHIV plasma viral loads. (A) Plasma viral loads in rhesus macaques that were left

666

untreated (control), or treated as described above. Each animal is represented by a distinct line.

667

The black arrows indicate day of mAb administration and the shaded areas correspond to the

668

duration of daily ART treatment. (B) Geometric mean plasma viral loads for the untreated and

669

the 3 treatment groups (n=6 per group). The error bars represent the 95% confidence interval at

670

each time point. Peak viremia (C), day 10-21 viremia area under the curve (D), and the slope of

671

viremia decay from peak to day 21 (E) are shown for each animal by treatment group with lines

672

at top indicating significant differences relative to untreated control animals (* p