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.

Amino Acid Polymorphisms on the Brazilian Strain of Yellow Fever Virus Methyltransferase Are Related to the Host's Immune Evasion Mediated by Type I Interferon.

Since late 2016, a yellow fever virus (YFV) variant carrying a set of nine amino acid variations has circulated in South America. Three of them were mapped on the methyltransferase (MTase) domain of viral NS5 protein. To assess whether these changes affected viral infectivity, we synthesized YFV carrying the MTase of circulating lineage as well as its isoform with the residues of the previous strains (NS5 K101R, NS5 V138I, and NS5 G173S). We observed a slight difference in viral growth properties and plaque phenotype between the two synthetic YFVs. However, the MTase polymorphisms associated with the Brazilian strain of YFV (2016-2019) confer more susceptibility to the IFN-I. In addition, in vitro MTase assay revealed that the interaction between the YFV MTase and the methyl donor molecule (SAM) is altered in the Brazilian MTase variant. Altogether, the results reported here describe that the MTase carrying the molecular signature of the Brazilian YFV circulating since 2016 might display a slight decrease in its catalytic activity but virtually no effect on viral fitness in the parameters comprised in this study. The most marked influence of these residues stands in the immune escape against the antiviral response mediated by IFN-I.

Genotype-specific features reduce the susceptibility of South American yellow fever virus strains to vaccine-induced antibodies.

The resurgence of yellow fever in South America has prompted vaccination against the etiologic agent, yellow fever virus (YFV). Current vaccines are based on a live-attenuated YF-17D virus derived from a virulent African isolate. The capacity of these vaccines to induce neutralizing antibodies against the vaccine strain is used as a surrogate for protection. However, the sensitivity of genetically distinct South American strains to vaccine-induced antibodies is unknown. We show that antiviral potency of the polyclonal antibody response in vaccinees is attenuated against an emergent Brazilian strain. This reduction was attributable to amino acid changes at two sites in central domain II of the glycoprotein E, including multiple changes at the domain I-domain II hinge, which are unique to and shared among most South American YFV strains. Our findings call for a reevaluation of current approaches to YFV immunological surveillance in South America and suggest approaches for updating vaccines.

Post-acute COVID-19 syndrome after reinfection and vaccine breakthrough by the SARS-CoV-2 Gamma variant in Brazil.

We describe a case of prolonged COVID-19 caused by the SARS-CoV-2 Gamma variant in a fully vaccinated healthcare worker, 387 days after an infection caused by lineage B.1.1.33. Infections were confirmed by whole-genome sequencing and corroborated by the detection of neutralizing antibodies in convalescent serum samples. Considering the permanent exposure of this healthcare worker to SARS-CoV-2, the waning immunity after the first infection, the low efficacy of the inactivated vaccine at preventing COVID-19, the immune escape of the Gamma variant (VOC), and the burden of post-COVID syndrome, this individual would have benefited from an additional dose of a heterologous vaccine.

ZIKA Virus Neutralizing Antibody Kinetics in Antenatally Exposed Infants.

BACKGROUND: Zika virus (ZIKV) is associated with severe congenital abnormalities and laboratory diagnosis of antenatal infection is difficult. Here we evaluated ZIKV neutralizing antibody (nAb) kinetics in infants born to mothers with PCR-confirmed ZIKV infection during pregnancy. METHODS: Neonates (n = 98) had serum specimens tested repeatedly for ZIKV nAb over the first 2 years of life using virus neutralization test (VNT). ZIKV neonatal infection was confirmed by RT-PCR in blood or urine and/or presence of ZIKV IgM antibodies, and results were correlated with infant clinical features. RESULTS: Postnatal laboratory evidence of ZIKV vertical transmission was obtained for 60.2% of children, while 32.7% exhibited clinical abnormalities. Congenital abnormalities were found in 37.3% of children with confirmed ZIKV infection and 31.0% of children without confirmed infection (P = .734). All but 1 child displayed a physiologic decline in ZIKV nAb, reflecting maternal antibody decay, despite an early ZIKV-IgM response in one-third of infants. CONCLUSIONS: Infants with antenatal ZIKV exposure do not develop ZIKV nAb despite an early IgM response. Therefore, ZIKV VNT in children is not useful for diagnosis of congenital infection. In light of these findings, it remains to be determined if children infected in utero are potentially susceptible to reinfection.

Immune outcomes of Zika virus infection in nonhuman primates.

Although the Zika virus (ZIKV) epidemic is subsiding, immune responses that are important for controlling acute infection have not been definitively characterized. Nonhuman primate (NHP) models were rapidly developed to understand the disease and to test vaccines, and these models have since provided an understanding of the immune responses that correlate with protection during natural infection and vaccination. Here, we infected a small group of male rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques with a minimally passaged Brazilian ZIKV isolate and used multicolor flow cytometry and transcriptional profiling to describe early immune patterns following infection. We found evidence of strong innate antiviral responses together with induction of neutralizing antibodies and T cell responses. We also assessed the relative importance of CD8 T cells in controlling infection by carrying out CD8 T cell depletion in an additional two animals of each species. CD8 depletion appeared to dysregulate early antiviral responses and possibly increase viral persistence, but the absence of CD8 T cells ultimately did not impair control of the virus. Together, these data describe immunological trends in two NHP species during acute ZIKV infection, providing an account of early responses that may be important in controlling infection.

Zika virus vertical transmission in children with confirmed antenatal exposure.

We report Zika virus (ZIKV) vertical transmission in 130 infants born to PCR+ mothers at the time of the Rio de Janeiro epidemic of 2015-2016. Serum and urine collected from birth through the first year of life were tested by quantitative reverse transcriptase polymerase chain reaction (PCR) and/or IgM Zika MAC-ELISA. Four hundred and seven specimens are evaluated; 161 sera tested by PCR and IgM assays, 85 urines by PCR. Sixty-five percent of children (N = 84) are positive in at least one assay. Of 94 children tested within 3 months of age, 70% are positive. Positivity declines to 33% after 3 months. Five children are PCR+ beyond 200 days of life. Concordance between IgM and PCR results is 52%, sensitivity 65%, specificity 40% (positive PCR results as gold standard). IgM and serum PCR are 61% concordant; serum and urine PCR 55%. Most children (65%) are clinically normal. Equal numbers of children with abnormal findings (29 of 45, 64%) and normal findings (55 of 85, 65%) have positive results, p = 0.98. Earlier maternal trimester of infection is associated with positive results (p = 0.04) but not clinical disease (p = 0.98). ZIKV vertical transmission is frequent but laboratory confirmed infection is not necessarily associated with infant abnormalities.

A Zika virus primary isolate induces neuroinflammation, compromises the blood-brain barrier and upregulates CXCL12 in adult macaques.

Zika virus (ZIKV) is a flavivirus that can cause neuropathogenesis in adults and fetal neurologic malformation following the infection of pregnant women. We used a nonhuman primate model, the Indian-origin Rhesus macaque (IRM), to gain insight into virus-associated hallmarks of ZIKV-induced adult neuropathology. We find that the virus causes prevalent acute and chronic neuroinflammation and chronic disruption of the blood-brain barrier (BBB) in adult animals. ZIKV infection resulted in specific short- and long-term augmented expression of the chemokine CXCL12 in the central nervous system (CNS)of adult IRMs. Moreover, CXCL12 expression persists long after the initial viral infection is apparently cleared. CXCL12 plays a key role both in regulating lymphocyte trafficking through the BBB to the CNS and in mediating repair of damaged neural tissue including remyelination. Understanding how CXCL12 expression is controlled will likely be of central importance in the definition of ZIKV-associated neuropathology in adults.

Zika virus targets the human thymic epithelium.

Previous work showed that the thymus can be infected by RNA viruses as HIV and HTLV-1. We thus hypothesized that the thymus might also be infected by the Zika virus (ZIKV). Herein we provide compelling evidence that ZIKV targets human thymic epithelial cells (TEC) in vivo and in vitro. ZIKV-infection enhances keratinization of TEC, with a decrease in proliferation and increase in cell death. Moreover, ZIKV modulates a high amount of coding RNAs with upregulation of genes related to cell adhesion and migration, as well as non-coding genes including miRNAs, circRNAs and lncRNAs. Moreover, we observed enhanced attachment of lymphoblastic T-cells to infected TEC, as well as virus transfer to those cells. Lastly, alterations in thymuses from babies congenitally infected were seen, with the presence of viral envelope protein in TEC. Taken together, our data reveals that the thymus, particularly the thymic epithelium, is a target for the ZIKV with changes in the expression of molecules that are relevant for interactions with developing thymocytes.

Low vector competence in sylvatic mosquitoes limits Zika virus to initiate an enzootic cycle in South America.

Zika virus (ZIKV) has spread in the Americas since 2015 and the potential establishment of a sylvatic transmission cycle in the continent has been hypothesized. We evaluated vector competence of five sylvatic Neotropical mosquito species to two ZIKV isolates. Distinct batches of Haemagogus leucoceleanus, Sabethes albiprivus, Sabethes identicus, Aedes terrens and Aedes scapularis females were respectively orally challenged and inoculated intrathoracically with ZIKV. Orally challenged mosquitoes were refractory or exhibited low infection rates. Viral dissemination was detected only in Hg. leucocelaenus, but with very low rates. Virus was not detected in saliva of any mosquito orally challenged with ZIKV, regardless of viral isolate and incubation time. When intrathoracically injected, ZIKV disseminated in high rates in Hg. leucocelaenus, Sa. identicus and Sa. albpiprivus, but low transmission was detected in these species; very low dissemination and no transmission was detected in Ae. terrens and Ae. scapularis. Together these results suggest that genetically determined tissue barriers, especially in the midgut, play a vital role in inhibiting ZIKV for transmission in the tested sylvatic mosquito species. Thus, an independent enzootic transmission cycle for ZIKV in South America is very unlikely.

Zika virus infects human blood mononuclear cells.

BACKGROUND: Zika virus (ZIKV) infection gained public health concern after the 2015 outbreak in Brazil, when microcephaly rates increased in babies born from infected mothers. It was demonstrated that ZIKV causes a congenital Zika virus syndrome, including various alterations in the development of the central nervous system. Although the infection of cells from the nervous system has been well documented, less is known in respect of ZIKV ability to infect immune cells. Herein, we investigated if peripheral blood mononuclear cells (PBMCs), freshly-isolated from healthy donors, could be infected by ZIKV. METHODS: PBMCs from healthy donors were isolated and cultured in medium with ZIKV strain Rio-U1 (MOI = 0.1). Infection was analyzed by RT-qPCR and flow cytometry. RESULTS: We detected the ZIKV RNA in PBMCs from all donors by RT-qPCR analysis. The detection of viral antigens by flow cytometry revealed that PBMC from more than 50% the donors were infected by ZIKV, with CD3+CD4+ T cells, CD3-CD19+ B cells and CD3+CD8+ T cells being, respectively, the most frequently infected subpopulations, followed by CD14+ monocytes. Additionally, we observed high variability in PBMC infection rates among different donors, either by numbers or type infected cells. CONCLUSIONS: These findings raise the hypothesis that PBMCs can act as a reservoir of the virus, which may facilitate viral dissemination to different organs, including immune-privileged sites.

Postnatal Zika virus infection of nonhuman primate infants born to mothers infected with homologous Brazilian Zika virus.

Recent data in a nonhuman primate model showed that infants postnatally infected with Zika virus (ZIKV) were acutely susceptible to high viremia and neurological damage, suggesting the window of vulnerability extends beyond gestation. In this pilot study, we addressed the susceptibility of two infant rhesus macaques born healthy to dams infected with Zika virus during pregnancy. Passively acquired neutralizing antibody titers dropped below detection limits between 2 and 3 months of age, while binding antibodies remained detectable until viral infection at 5 months. Acute serum viremia was comparatively lower than adults infected with the same Brazilian isolate of ZIKV (n = 11 pregnant females, 4 males, and 4 non-pregnant females). Virus was never detected in cerebrospinal fluid nor in neural tissues at necropsy two weeks after infection. However, viral RNA was detected in lymph nodes, confirming some tissue dissemination. Though protection was not absolute and our study lacks an important comparison with postnatally infected infants born to naïve dams, our data suggest infants born healthy to infected mothers may harbor a modest but important level of protection from postnatally acquired ZIKV for several months after birth, an encouraging result given the potentially severe infection outcomes of this population.

Mamu-B*17+ Rhesus Macaques Vaccinated with env, vif, and nef Manifest Early Control of SIVmac239 Replication.

Certain major histocompatibility complex class I (MHC-I) alleles are associated with spontaneous control of viral replication in human immunodeficiency virus (HIV)-infected people and simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs). These cases of "elite" control of HIV/SIV replication are often immune-mediated, thereby providing a framework for studying anti-lentiviral immunity. In this study, we examined how vaccination impacts SIV replication in RMs expressing the MHC-I allele Mamu-B*17 Approximately 21% of Mamu-B*17+ and 50% of Mamu-B*08+ RMs control chronic-phase viremia after SIVmac239 infection. Because CD8+ T cells targeting Mamu-B*08-restricted SIV epitopes have been implicated in virologic suppression in Mamu-B*08+ RMs, we investigated whether this might also be true for Mamu-B*17+ RMs. Two groups of Mamu-B*17+ RMs were vaccinated with genes encoding Mamu-B*17-restricted epitopes in Vif and Nef. These genes were delivered by themselves (group 1) or together with env (group 2). Group 3 included MHC-I-matched RMs and served as the control group. Surprisingly, the group 1 vaccine regimen had little effect on viral replication compared to group 3, suggesting that unlike Mamu-B*08+ RMs, preexisting SIV-specific CD8+ T cells alone do not facilitate long-term virologic suppression in Mamu-B*17+ RMs. Remarkably, however, 5/8 group 2 vaccinees controlled viremia to <15 viral RNA copies/ml soon after infection. No serological neutralizing activity against SIVmac239 was detected in group 2, although vaccine-elicited gp140-binding antibodies correlated inversely with nadir viral loads. Collectively, these data shed new light on the unique mechanism of elite control in Mamu-B*17+ RMs and implicate vaccine-induced, nonneutralizing anti-Env antibodies in the containment of immunodeficiency virus infection.IMPORTANCE A better understanding of the immune correlates of protection against HIV might facilitate the development of a prophylactic vaccine. Therefore, we investigated simian immunodeficiency virus (SIV) infection outcomes in rhesus macaques expressing the major histocompatibility complex class I allele Mamu-B*17 Approximately 21% of Mamu-B*17+ macaques spontaneously controlled chronic phase viremia after SIV infection, an effect that may involve CD8+ T cells targeting Mamu-B*17-restricted SIV epitopes. We vaccinated Mamu-B*17+ macaques with genes encoding immunodominant epitopes in Vif and Nef alone (group 1) or together with env (group 2). Although neither vaccine regimen prevented SIV infection, 5/8 group 2 vaccinees controlled viremia to below detection limits shortly after infection. This outcome, which was not observed in group 1, was associated with vaccine-induced, nonneutralizing Env-binding antibodies. Together, these findings suggest a limited contribution of Vif- and Nef-specific CD8+ T cells for virologic control in Mamu-B*17+ macaques and implicate anti-Env antibodies in containment of SIV infection.

Fetal demise and failed antibody therapy during Zika virus infection of pregnant macaques.

Zika virus (ZIKV) infection of pregnant women is associated with pathologic complications of fetal development. Here, we infect pregnant rhesus macaques (Macaca mulatta) with a minimally passaged ZIKV isolate from Rio de Janeiro, where a high rate of fetal development complications was observed. The infection of pregnant macaques with this virus results in maternal viremia, virus crossing into the amniotic fluid (AF), and in utero fetal deaths. We also treated three additional ZIKV-infected pregnant macaques with a cocktail of ZIKV-neutralizing human monoclonal antibodies (nmAbs) at peak viremia. While the nmAbs can be effective in clearing the virus from the maternal sera of treated monkeys, it is not sufficient to clear ZIKV from AF. Our report suggests that ZIKV from Brazil causes fetal demise in non-human primates (NHPs) without additional mutations or confounding co-factors. Treatment with a neutralizing anti-ZIKV nmAb cocktail is insufficient to fully stop vertical transmission.

Zika virus can be venereally transmitted between Aedes aegypti mosquitoes.

BACKGROUND: Alternative transmission routes have been described for Zika virus (ZIKV). Here, we assessed for the first time the venereal transmission of ZIKV between Aedes aegypti under laboratory conditions. RESULTS: Orally-infected mosquito females were able to transmit the virus to males venereally, and males inoculated intrathoracically were capable of infecting females during mating. The genome of venereally-transmitted virus recovered from males was identical to that of ZIKV ingested by mated females. CONCLUSION: We conclude that venereal transmission between Aedes mosquitoes might contribute to Zika virus maintenance in nature.

Neutralizing human monoclonal antibodies prevent Zika virus infection in macaques.

Therapies to prevent maternal Zika virus (ZIKV) infection and its subsequent fetal developmental complications are urgently required. We isolated three potent ZIKV-neutralizing monoclonal antibodies (nmAbs) from the plasmablasts of a ZIKV-infected patient-SMZAb1, SMZAb2, and SMZAb5-directed against two different domains of the virus. We engineered these nmAbs with Fc LALA mutations that abrogate Fcγ receptor binding, thus eliminating potential therapy-mediated antibody-dependent enhancement. We administered a cocktail of these three nmAbs to nonhuman primates 1 day before challenge with ZIKV and demonstrated that the nmAbs completely prevented viremia in serum after challenge. Given that numerous antibodies have exceptional safety profiles in humans, the cocktail described here could be rapidly developed to protect uninfected pregnant women and their fetuses.

Culex quinquefasciatus from areas with the highest incidence of microcephaly associated with Zika virus infections in the Northeast Region of Brazil are refractory to the virus.

Zika virus (ZIKV) is widely distributed in Brazil and the Northeast Region (NE) is the most affected zone, showing the highest incidence of microcephaly associated with ZIKV congenital infections worldwide. We report attempts to infect three populations of Culex quinquefasciatus from severely affected sites in the NE and Southeast Region (SE) of Brazil with three strains of ZIKV isolated from these localities. An Aedes aegypti population from the SE was used as a positive control. All tested Cx. quinquefasciatus populations were refractory to the ZIKV isolates. For these reasons, we believe Cx. quinquefasciatus should not be considered a potential vector of ZIKV in Brazil.

Culex quinquefasciatus from areas with the highest incidence of microcephaly associated with Zika virus infections in the Northeast Region of Brazil are refractory to the virus

Zika virus (ZIKV) is widely distributed in Brazil and the Northeast Region (NE) is the most affected zone, showing the highest incidence of microcephaly associated with ZIKV congenital infections worldwide. We report attempts to infect three populations of Culex quinquefasciatus from severely affected sites in the NE and Southeast Region (SE) of Brazil with three strains of ZIKV isolated from these localities. An Aedes aegypti population from the SE was used as a positive control. All tested Cx. quinquefasciatus populations were refractory to the ZIKV isolates. For these reasons, we believe Cx. quinquefasciatus should not be considered a potential vector of ZIKV in Brazil.