Therapeutic neutralizing monoclonal antibody administration protects against lethal yellow fever virus infection.

Yellow fever virus (YFV) is a reemerging global health threat, driven by several factors, including increased spread of the mosquito vector and rapid urbanization. Although a prophylactic vaccine exists, vaccine hesitancy, supply deficits, and distribution difficulties leave specific populations at risk of severe YFV disease, as evidenced by recent outbreaks in South America. To establish a treatment for patients with severe YFV infection, we tested 37 YFV-specific monoclonal antibodies isolated from vaccinated humans and identified two capable of potently neutralizing multiple pathogenic primary YFV isolates. Using both hamster and nonhuman primate models of lethal YFV infection, we demonstrate that a single administration of either of these two potently neutralizing antibodies during acute infection fully controlled viremia and prevented severe disease and death in treated animals. Given the potential severity of YFV-induced disease, our results show that these antibodies could be effective in saving lives and fill a much-needed void in managing YFV cases during outbreaks.

Molecular insights into antibody-mediated protection against the prototypic simian immunodeficiency virus.

SIVmac239 infection of macaques is a favored model of human HIV infection. However, the SIVmac239 envelope (Env) trimer structure, glycan occupancy, and the targets and ability of neutralizing antibodies (nAbs) to protect against SIVmac239 remain unknown. Here, we report the isolation of SIVmac239 nAbs that recognize a glycan hole and the V1/V4 loop. A high-resolution structure of a SIVmac239 Env trimer-nAb complex shows many similarities to HIV and SIVcpz Envs, but with distinct V4 features and an extended V1 loop. Moreover, SIVmac239 Env has a higher glycan shield density than HIV Env that may contribute to poor or delayed nAb responses in SIVmac239-infected macaques. Passive transfer of a nAb protects macaques from repeated intravenous SIVmac239 challenge at serum titers comparable to those described for protection of humans against HIV infection. Our results provide structural insights for vaccine design and shed light on antibody-mediated protection in the SIV model.

Non-neutralizing Antibodies May Contribute to Suppression of SIVmac239 Viremia in Indian Rhesus Macaques.

The antiviral properties of broadly neutralizing antibodies against HIV are well-documented but no vaccine is currently able to elicit protective titers of these responses in primates. While current vaccine modalities can readily induce non-neutralizing antibodies against simian immunodeficiency virus (SIV) and HIV, the ability of these responses to restrict lentivirus transmission and replication remains controversial. Here, we investigated the antiviral properties of non-neutralizing antibodies in a group of Indian rhesus macaques (RMs) that were vaccinated with vif, rev, tat, nef, and env, as part of a previous study conducted by our group. These animals manifested rapid and durable control of viral replication to below detection limits shortly after SIVmac239 infection. Although these animals had no serological neutralizing activity against SIVmac239 prior to infection, their pre-challenge titers of Env-binding antibodies correlated with control of viral replication. To assess the contribution of anti-Env humoral immune responses to virologic control in two of these animals, we transiently depleted their circulating antibodies via extracorporeal plasma immunoadsorption and inhibition of IgG recycling through antibody-mediated blockade of the neonatal Fc receptor. These procedures reduced Ig serum concentrations by up to 80% and temporarily induced SIVmac239 replication in these animals. Next, we transferred purified total Ig from the rapid controllers into six vaccinated RMs one day before intrarectal challenge with SIVmac239. Although recipients of the hyperimmune anti-SIV Ig fraction were not protected from infection, their peak and chronic phase viral loads were significantly lower than those in concurrent unvaccinated control animals. Together, our results suggest that non-neutralizing Abs may play a role in the suppression of SIVmac239 viremia.

Rhesus Cytomegalovirus-Specific CD8+ Cytotoxic T Lymphocytes Do Not Become Functionally Exhausted in Chronic SIVmac239 Infection.

CD8+ cytotoxic T lymphocytes (CTLs) exert potent antiviral activity after HIV/SIV infection. However, efforts to harness the antiviral efficacy of CTLs for HIV/SIV prophylaxis and therapy have been severely hindered by two major problems: viral escape and exhaustion. By contrast, CTLs directed against human cytomegalovirus (HCMV), a ubiquitous chronic herpesvirus, seldom select for escape mutations and remain functional and refractory to exhaustion during chronic HCMV and HIV infection. Recently, attempts have been made to retarget HCMV-specific CTLs for cancer immunotherapy. We speculate that such a strategy may also be beneficial in the context of HIV/SIV infection, facilitating CTL-mediated control of HIV/SIV replication. As a preliminary assessment of the validity of this approach, we investigated the phenotypes and functionality of rhesus CMV (RhCMV)-specific CTLs in SIVmac239-infected Indian rhesus macaques (RMs), a crucial HIV animal model system. We recently identified two immunodominant, Mamu-A∗02-restricted CTL epitopes derived from RhCMV proteins and sought to evaluate the phenotypic and functional characteristics of these CTL populations in chronic SIVmac239 infection. We analyzed and directly compared RhCMV- and SIVmac239-specific CTLs during SIVmac239 infection in a cohort of Mamu-A∗01+ and Mamu-A∗02+ RMs. CTL populations specific for at least one of the RhCMV-derived CTL epitopes were detected in ten of eleven Mamu-A∗02+ animals tested, and both populations were detected in five of these animals. Neither RhCMV-specific CTL population exhibited significant changes in frequency, memory phenotype, granzyme B expression, exhaustion marker (PD-1 and CTLA-4) expression, or polyfunctionality between pre- and chronic SIVmac239 infection timepoints. In chronic SIVmac239 infection, RhCMV-specific CTLs exhibited higher levels of granzyme B expression and polyfunctionality, and lower levels of exhaustion marker expression, than SIVmac239-specific CTLs. Additionally, compared to SIVmac239-specific CTLs, greater proportions of RhCMV-specific CTLs were of the terminally differentiated effector memory phenotype (CD28- CCR7-) during chronic SIVmac239 infection. These results suggest that, in contrast to SIVmac239-specific CTLs, RhCMV-specific CTLs maintain their phenotypes and cytolytic effector functions during chronic SIVmac239 infection, and that retargeting RhCMV-specific CTLs might be a promising SIV immunotherapeutic strategy.

An Automated Fluorescence-Based Method to Isolate Bone Marrow-Derived Plasma Cells from Rhesus Macaques Using SIVmac239 SOSIP.664.

Simian immunodeficiency virus (SIV) infection of Indian rhesus macaques (RMs) is one of the best-characterized animal models for human immunodeficiency virus (HIV) infection. Monoclonal antibodies (mAbs) have shown promise for prevention and treatment of HIV infection. However, it has been difficult to isolate mAbs that potently neutralize the highly pathogenic SIVmac239 strain. This has been largely due to the low frequency of circulating B cells encoding neutralizing Abs. Here we describe a novel technique to isolate mAbs directly from bone marrow-derived, Ab-secreting plasma cells. We employed an automated micromanipulator to isolate single SIVmac239 SOSIP.664-specific plasma cells from the bone marrow of a SIVmac239-infected RM with serum neutralization titers against SIVmac239. After picking plasma cells, we obtained 44 paired Ab sequences. Ten of these mAbs were SIV specific. Although none of these mAbs neutralized SIVmac239, three mAbs completely neutralized the related SIVmac316 strain. The majority of these mAbs bound to primary rhesus CD4+ T cells infected with SIVmac239 and induced Ab-dependent cellular cytotoxicity. This method is a first step in successful isolation of antigen-specific bone marrow-derived plasma cells from RMs.

Induction of Transient Virus Replication Facilitates Antigen-Independent Isolation of SIV-Specific Monoclonal Antibodies.

Structural characterization of the HIV-1 Envelope (Env) glycoprotein has facilitated the development of Env probes to isolate HIV-specific monoclonal antibodies (mAbs). However, preclinical studies have largely evaluated these virus-specific mAbs against chimeric viruses, which do not naturally infect non-human primates, in contrast to the unconstrained simian immunodeficiency virus (SIV)mac239 clone. Given the paucity of native-like reagents for the isolation of SIV-specific B cells, we examined a method to isolate SIVmac239-specific mAbs without using Env probes. We first activated virus-specific B cells by inducing viral replication after the infusion of a CD8ß-depleting mAb or withdrawal of antiretroviral therapy in SIVmac239-infected rhesus macaques. Following the rise in viremia, we observed 2- to 4-fold increases in the number of SIVmac239 Env-reactive plasmablasts in circulation. We then sorted these activated B cells and obtained 206 paired Ab sequences. After expressing 122 mAbs, we identified 14 Env-specific mAbs. While these Env-specific mAbs bound to both the SIVmac239 SOSIP.664 trimer and to infected primary rhesus CD4+ T cells, five also neutralized SIVmac316. Unfortunately, none of these mAbs neutralized SIVmac239. Our data show that this method can be used to isolate virus-specific mAbs without antigenic probes by inducing bursts of contemporary replicating viruses in vivo.

A human inferred germline antibody binds to an immunodominant epitope and neutralizes Zika virus.

The isolation of neutralizing monoclonal antibodies (nmAbs) against the Zika virus (ZIKV) might lead to novel preventative strategies for infections in at-risk individuals, primarily pregnant women. Here we describe the characterization of human mAbs from the plasmablasts of an acutely infected patient. One of the 18 mAbs had the unusual feature of binding to and neutralizing ZIKV despite not appearing to have been diversified by affinity maturation. This mAb neutralized ZIKV (Neut50 ~ 2 µg/ml) but did not react with any of the four dengue virus serotypes. Except for the expected junctional diversity created by the joining of the V-(D)-J genes, there was no deviation from immunoglobulin germline genes. This is a rare example of a human mAb with neutralizing activity in the absence of detectable somatic hypermutation. Importantly, binding of this mAb to ZIKV was specifically inhibited by human plasma from ZIKV-exposed individuals, suggesting that it may be of value in a diagnostic setting.

Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a first-line tuberculosis (TB) drug that has been in clinical use for 60 years yet still has an unresolved mechanism of action. Based upon the observation that the minimum concentration of PZA required to inhibit the growth of Mycobacterium tuberculosis is approximately 1,000-fold higher than that of other first-line drugs, we hypothesized that M. tuberculosis expresses factors that mediate intrinsic resistance to PZA. To identify genes associated with intrinsic PZA resistance, a library of transposon-mutagenized Mycobacterium bovis BCG strains was screened for strains showing hypersusceptibility to the active form of PZA, pyrazinoic acid (POA). Disruption of the long-chain fatty acyl coenzyme A (CoA) ligase FadD2 enhanced POA susceptibility by 16-fold on agar medium, and the wild-type level of susceptibility was restored upon expression of fadD2 from an integrating mycobacterial vector. Consistent with the recent observation that POA perturbs mycobacterial CoA metabolism, the fadD2 mutant strain was more vulnerable to POA-mediated CoA depletion than the wild-type strain. Ectopic expression of the M. tuberculosis pyrazinamidase PncA, necessary for conversion of PZA to POA, in the fadD2 transposon insertion mutant conferred at least a 16-fold increase in PZA susceptibility under active growth conditions in liquid culture at neutral pH. Importantly, deletion of fadD2 in M. tuberculosis strain H37Rv also resulted in enhanced susceptibility to POA. These results indicate that FadD2 is associated with intrinsic PZA and POA resistance and provide a proof of concept for the target-based potentiation of PZA activity in M. tuberculosis.

Uncoupling Environmental pH and Intrabacterial Acidification from Pyrazinamide Susceptibility in Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a first-line antitubercular drug for which the mode of action remains unresolved. Mycobacterium tuberculosis lacks measurable susceptibility to PZA under standard laboratory growth conditions. However, susceptibility to this drug can be induced by cultivation of the bacilli in an acidified growth medium. Previous reports suggested that the active form of PZA, pyrazinoic acid (POA), operates as a proton ionophore that confers cytoplasmic acidification when M. tuberculosis is exposed to an acidic environment. In this study, we demonstrate that overexpression of the PZA-activating enzyme PncA can confer PZA susceptibility to M. tuberculosis under neutral and even alkaline growth conditions. Furthermore, we find that wild-type M. tuberculosis displays increased susceptibility to POA relative to PZA in neutral and alkaline media. Utilizing a strain of M. tuberculosis that expresses a pH-sensitive green fluorescent protein (GFP), we find that unlike the bona fide ionophores monensin and carbonyl cyanide 3-chlorophenylhydrazone, PZA and POA do not induce rapid uncoupling or cytoplasmic acidification under conditions that promote susceptibility. Thus, based on these observations, we conclude that the antitubercular action of POA is independent of environmental pH and intrabacterial acidification.

Pantothenate and pantetheine antagonize the antitubercular activity of pyrazinamide.

Pyrazinamide (PZA) is a first-line tuberculosis drug that inhibits the growth of Mycobacterium tuberculosis via an as yet undefined mechanism. An M. tuberculosis laboratory strain that was auxotrophic for pantothenate was found to be insensitive to PZA and to the active form, pyrazinoic acid (POA). To determine whether this phenotype was strain or condition specific, the effect of pantothenate supplementation on PZA activity was assessed using prototrophic strains of M. tuberculosis. It was found that pantothenate and other ß-alanine-containing metabolites abolished PZA and POA susceptibility, suggesting that POA might selectively target pantothenate synthesis. However, when the pantothenate-auxotrophic strain was cultivated using a subantagonistic concentration of pantetheine in lieu of pantothenate, susceptibility to PZA and POA was restored. In addition, we found that ß-alanine could not antagonize PZA and POA activity against the pantothenate-auxotrophic strain, indicating that the antagonism is specific to pantothenate. Moreover, pantothenate-mediated antagonism was observed for structurally related compounds, including n-propyl pyrazinoate, 5-chloropyrazinamide, and nicotinamide, but not for nicotinic acid or isoniazid. Taken together, these data demonstrate that while pantothenate can interfere with the action of PZA, pantothenate synthesis is not directly targeted by PZA. Our findings suggest that targeting of pantothenate synthesis has the potential to enhance PZA efficacy and possibly to restore PZA susceptibility in isolates with panD-linked resistance.