Improved delivery of broadly neutralizing antibodies by nanocapsules suppresses SHIV infection in the CNS of infant rhesus macaques.

Broadly neutralizing antibodies (bNAbs) directed to HIV-1 have shown promise at suppressing viremia in animal models. However, the use of bNAbs for the central nervous system (CNS) infection is confounded by poor penetration of the blood brain barrier (BBB). Typically, antibody concentrations in the CNS are extremely low; with levels in cerebrospinal fluid (CSF) only 0.1% of blood concentrations. Using a novel nanotechnology platform, which we term nanocapsules, we show effective transportation of the human bNAb PGT121 across the BBB in infant rhesus macaques upon systemic administration up to 1.6% of plasma concentration. We demonstrate that a single dose of PGT121 encased in nanocapsules when delivered at 48h post-infection delays early acute infection with SHIVSF162P3 in infants, with one of four animals demonstrating viral clearance. Importantly, the nanocapsule delivery of PGT121 improves suppression of SHIV infection in the CNS relative to controls.

Protection of Newborn Macaques by Plant-Derived HIV Broadly Neutralizing Antibodies: a Model for Passive Immunotherapy during Breastfeeding.

Preventing human immunodeficiency virus (HIV) infection in newborns by vertical transmission remains an important unmet medical need in resource-poor areas where antiretroviral therapy (ART) is not available and mothers and infants cannot be treated prepartum or during the breastfeeding period. In the present study, the protective efficacy of the potent HIV-neutralizing antibodies PGT121 and VRC07-523, both produced in plants, were assessed in a multiple-SHIV (simian-human immunodeficiency virus)-challenge breastfeeding macaque model. Newborn macaques received either six weekly subcutaneous injections with PGT121 alone or as a cocktail of PGT121-LS plus VRC07-523-LS injected three times every 2 weeks. Viral challenge with SHIVSF162P3 was twice weekly over 5.5 weeks using 11 exposures. Despite the transient presence of plasma viral RNA either immediately after the first challenge or as single-point blips, the antibodies prevented a productive infection in all babies with no sustained plasma viremia, compared to viral loads ranging from 103 to 5 × 108 virions/ml in four untreated controls. No virus was detected in peripheral blood mononuclear cells (PBMCs), and only 3 of 159 tissue samples were weakly positive in the treated babies. Newborn macaques proved to be immunocompetent, producing transient anti-Env antibodies and anti-drug antibody (ADA), which were maintained in the circulation after passive broadly neutralizing antibody clearance. ADA responses were directed to the IgG1 Fc CH2-CH3 domains, which has not been observed to date in adult monkeys passively treated with PGT121 or VRC01. In addition, high levels of VRC07-523 anti-idiotypic antibodies in the circulation of one newborn was concomitant with the rapid elimination of VRC07. Plant-expressed antibodies show promise as passive immunoprophylaxis in a breastfeeding model in newborns. IMPORTANCE Plant-produced human neutralizing antibody prophylaxis is highly effective in preventing infection in newborn monkeys during repeated oral exposure, modeling virus in breastmilk, and offers advantages in cost of production and safety. These findings raise the possibility that anti-Env antibodies may contribute to the control of viral replication in this newborn model and that the observed immune responsiveness may be driven by the long-lived presence of immune complexes.

Polyfunctional Tier 2-Neutralizing Antibodies Cloned following HIV-1 Env Macaque Immunization Mirror Native Antibodies in a Human Donor.

Vaccine efforts to combat HIV are challenged by the global diversity of viral strains and shielding of neutralization epitopes on the viral envelope glycoprotein trimer. Even so, the isolation of broadly neutralizing Abs from infected individuals suggests the potential for eliciting protective Abs through vaccination. This study reports a panel of 58 mAbs cloned from a rhesus macaque (Macaca mulatta) immunized with envelope glycoprotein immunogens curated from an HIV-1 clade C-infected volunteer. Twenty mAbs showed neutralizing activity, and the strongest neutralizer displayed 92% breadth with a median IC50 of 1.35 µg/ml against a 13-virus panel. Neutralizing mAbs predominantly targeted linear epitopes in the V3 region in the cradle orientation (V3C) with others targeting the V3 ladle orientation (V3L), the CD4 binding site (CD4bs), C1, C4, or gp41. Nonneutralizing mAbs bound C1, C5, or undetermined conformational epitopes. Neutralization potency strongly correlated with the magnitude of binding to infected primary macaque splenocytes and to the level of Ab-dependent cellular cytotoxicity, but did not predict the degree of Ab-dependent cellular phagocytosis. Using an individualized germline gene database, mAbs were traced to 23 of 72 functional IgHV alleles. Neutralizing V3C Abs displayed minimal nucleotide somatic hypermutation in the H chain V region (3.77%), indicating that relatively little affinity maturation was needed to achieve in-clade neutralization breadth. Overall, this study underscores the polyfunctional nature of vaccine-elicited tier 2-neutralizing V3 Abs and demonstrates partial reproduction of the human donor's humoral immune response through nonhuman primate vaccination.

An HIV Vaccine Targeting the V2 Region of the HIV Envelope Induces a Highly Durable Polyfunctional Fc-Mediated Antibody Response in Rhesus Macaques.

The HIV vaccine field now recognizes the potential importance of generating polyfunctional antibodies (Abs). The only clinical HIV vaccine trial to date to show significant efficacy (RV144) found that reduced infection rates correlated with the level of nonneutralizing Abs specific for the V2 region of the envelope glycoprotein. We have conducted a comprehensive preclinical reverse vaccinology-based vaccine program that has included the design and production and testing of numerous scaffolded V2 region immunogens. The most immunogenic vaccine regimen in nonhuman primates among those studied as part of this program consisted of a cocktail of three immunogens presenting V2 from different viruses and clades in the context of different scaffolds. Presently we demonstrate that the V2-specific Ab response from this regimen was highly durable and functionally diverse for the duration of the study (25 weeks after the final immunization). The total IgG binding response at this late time point exhibited only an ∼5× reduction in potency. Three immunizations appeared essential for the elicitation of a strong Ab-dependent cellular cytotoxicity (ADCC) response for all animals, as opposed to the Ab-dependent cellular phagocytosis (ADCP) and virus capture responses, which were comparably potent after only 2 immunizations. All functionalities measured were highly durable through the study period. Therefore, testing this vaccine candidate for its protective capacity is warranted.IMPORTANCE The only HIV vaccine trial for which protective efficacy was detected correlated this efficacy with V2-specific Abs that were effectively nonneutralizing. This result has fueled a decade of HIV vaccine research focused on designing an HIV vaccine capable of eliciting V2-focused, polyfunctional Abs that effectively bind HIV and trigger various leukocytes to kill the virus and restrict viral spread. From the numerous vaccine candidates designed and tested as part of our V2-focused preclinical vaccine program, we have identified immunogens and a vaccine regimen that induces a highly durable and polyfunctional V2-focused Ab response in rhesus macaques, described herein.

Single-dose bNAb cocktail or abbreviated ART post-exposure regimens achieve tight SHIV control without adaptive immunity.

Vertical transmission accounts for most human immunodeficiency virus (HIV) infection in children, and treatments for newborns are needed to abrogate infection or limit disease progression. We showed previously that short-term broadly neutralizing antibody (bNAb) therapy given 24 h after oral exposure cleared simian-human immunodeficiency virus (SHIV) in a macaque model of perinatal infection. Here, we report that all infants given either a single dose of bNAbs at 30 h, or a 21-day triple-drug ART regimen at 48 h, are aviremic with almost no virus in tissues. In contrast, bNAb treatment beginning at 48 h leads to tight control without adaptive immune responses in half of animals. We conclude that both bNAbs and ART mediate effective post-exposure prophylaxis in infant macaques within 30-48 h of oral SHIV exposure. Our findings suggest that optimizing the treatment regimen may extend the window of opportunity for preventing perinatal HIV infection when treatment is delayed.

Multimeric Epitope-Scaffold HIV Vaccines Target V1V2 and Differentially Tune Polyfunctional Antibody Responses.

The V1V2 region of the HIV-1 envelope is the target of several broadly neutralizing antibodies (bNAbs). Antibodies to V1V2 elicited in the RV144 clinical trial correlated with a reduced risk of HIV infection, but these antibodies were without broad neutralizing activity. Antibodies targeting V1V2 also correlated with a reduced viral load in immunized macaques challenged with simian immunodeficiency virus (SIV) or simian/human immunodeficiency virus (SHIV). To focus immune responses on V1V2, we engrafted the native, glycosylated V1V2 domain onto five different multimeric scaffold proteins and conducted comparative immunogenicity studies in macaques. Vaccinated macaques developed high titers of plasma and mucosal antibodies that targeted structurally distinct V1V2 epitopes. Plasma antibodies displayed limited neutralizing activity but were functionally active for ADCC and phagocytosis, which was detectable 1-2 years after immunizations ended. This study demonstrates that multivalent, glycosylated V1V2-scaffold protein immunogens focus the antibody response on V1V2 and are differentially effective at inducing polyfunctional antibodies with characteristics associated with protection.

Reduced Cell-Associated DNA and Improved Viral Control in Macaques following Passive Transfer of a Single Anti-V2 Monoclonal Antibody and Repeated Simian/Human Immunodeficiency Virus Challenges.

A high level of V1V2-specific IgG antibodies (Abs) in vaccinees' sera was the only independent variable that correlated with a reduced risk of human immunodeficiency virus (HIV) acquisition in the RV144 clinical trial. In contrast, IgG avidity, antibody neutralization, and antibody-dependent cellular cytotoxicity each failed as independent correlates of infection. Extended analyses of RV144 samples demonstrated the antiviral activities of V1V2-specific vaccine-induced antibodies. V2-specific antibodies have also been associated with protection from simian immunodeficiency virus (SIV), and the V2i-specific subset of human monoclonal antibodies (MAbs), while poor neutralizers, mediates Fc-dependent antiviral functions in vitro The objective of this study was to determine the protective efficacy of a V2i-specific human MAb, 830A, against mucosal simian/human immunodeficiency virus (SHIV) challenge. V2i MAb binding sites overlap the integrin binding site in the V2 region and are similar to the epitopes bound by antibodies associated with reduced HIV infection rates in RV144. Because the IgG3 subclass was a correlate of reduced infection rates in RV144, we compared passive protection by both IgG1 and IgG3 subclasses of V2i MAb 830A. This experiment represents the first in vivo test of the hypothesis emanating from RV144 and SIV studies that V2i Abs can reduce the risk of infection. The results show that passive transfer with a single V2i MAb, IgG1 830A, reduced plasma and peripheral blood mononuclear cell (PBMC) virus levels and decreased viral DNA in lymphoid tissues compared to controls, but too few animals remained uninfected to achieve significance in reducing the risk of infection. Based on these findings, we conclude that V2i antibodies can impede virus seeding following mucosal challenge, resulting in improved virus control.IMPORTANCE Since the results of the HIV RV144 clinical trial were reported, there has been significant interest in understanding how protection was mediated. Antibodies directed to a subregion of the envelope protein called V1V2 were directly correlated with a reduced risk, and surprisingly low virus neutralization was observed. To determine whether these antibodies alone could mediate protection, we used a human monoclonal antibody directed to V2 with properties similar to those elicited in the vaccine trial for passive infusions in rhesus macaques and challenge with SHIV. The single V2 antibody at the dose given did not significantly reduce the number of infections, but there was a significant reduction in the seeding of virus to the lymph nodes and a decrease in plasma viremia in the HIV antibody-infused macaques compared with the control antibody-infused animals. This finding shows that V2 antibodies mediate antiviral activities in vivo that could contribute to a protective HIV vaccine.

Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in infant macaques.

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.