Forging Collaborative Relationships in Brazil: From AIDS to ZIKV.

The Zika virus (ZIKV) outbreak has stimulated collaborations between Brazilians, researchers from other South American countries, and scientists from around the world. The Brazilian response to the HIV/AIDS epidemic demonstrates capabilities that can be applied to the study of ZIKV and provides lessons for developing effective international infectious disease research collaborations.

SARS-CoV-2 Omicron virus causes attenuated disease in mice and hamsters.

The recent emergence of B.1.1.529, the Omicron variant1,2, has raised concerns of escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in preclinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of several B.1.1.529 isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. Despite modelling data indicating that B.1.1.529 spike can bind more avidly to mouse ACE2 (refs. 3,4), we observed less infection by B.1.1.529 in 129, C57BL/6, BALB/c and K18-hACE2 transgenic mice than by previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease and pathology with B.1.1.529 were also milder than with historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from the SAVE/NIAID network with several B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

Vaccine-induced systemic and mucosal T cell immunity to SARS-CoV-2 viral variants.

The first-generation COVID-19 vaccines have been effective in mitigating severe illness and hospitalization, but recurring waves of infections are associated with the emergence of SARS-CoV-2 variants that display progressive abilities to evade antibodies, leading to diminished vaccine effectiveness. The lack of clarity on the extent to which vaccine-elicited mucosal or systemic memory T cells protect against such antibody-evasive SARS-CoV-2 variants remains a critical knowledge gap in our quest for broadly protective vaccines. Using adjuvanted spike protein­based vaccines that elicit potent T cell responses, we assessed whether systemic or lung-resident CD4 and CD8 T cells protected against SARS-CoV-2 variants in the presence or absence of virus-neutralizing antibodies. We found that 1) mucosal or parenteral immunization led to effective viral control and protected against lung pathology with or without neutralizing antibodies, 2) protection afforded by mucosal memory CD8 T cells was largely redundant in the presence of antibodies that effectively neutralized the challenge virus, and 3) "unhelped" mucosal memory CD8 T cells provided no protection against the homologous SARS-CoV-2 without CD4 T cells and neutralizing antibodies. Significantly, however, in the absence of detectable virus-neutralizing antibodies, systemic or lung-resident memory CD4 and "helped" CD8 T cells provided effective protection against the relatively antibody-resistant B1.351 (ß) variant, without lung immunopathology. Thus, induction of systemic and mucosal memory T cells directed against conserved epitopes might be an effective strategy to protect against SARS-CoV-2 variants that evade neutralizing antibodies. Mechanistic insights from this work have significant implications in the development of T cell­targeted immunomodulation or broadly protective SARS-CoV-2 vaccines.

Molecular Surveillance Detects High Prevalence of the Neglected Parasite Mansonella ozzardi in the Colombian Amazon.

BACKGROUND: Mansonellosis is an undermapped insect-transmitted disease caused by filarial nematodes that are estimated to infect hundreds of millions of people. Despite their prevalence, there are many outstanding questions regarding the general biology and health impacts of the responsible parasites. Historical reports suggest that the Colombian Amazon is endemic for mansonellosis and may serve as an ideal location to pursue these questions. METHODS: We deployed molecular and classical approaches to survey Mansonella prevalence among adults belonging to indigenous communities along the Amazon River and its tributaries near Leticia, Colombia. RESULTS: Loop-mediated isothermal amplification (LAMP) assays on whole-blood samples detected a much higher prevalence of Mansonella ozzardi infection (approximately 40%) compared to blood smear microscopy or LAMP performed using plasma, likely reflecting greater sensitivity and the ability to detect low microfilaremias and occult infections. Mansonella infection rates increased with age and were higher among men. Genomic analysis confirmed the presence of M. ozzardi that clusters closely with strains sequenced in neighboring countries. We successfully cryopreserved M. ozzardi microfilariae, advancing the prospects of rearing infective larvae in controlled settings. CONCLUSION: These data suggest an underestimation of true mansonellosis prevalence, and we expect that these methods will help facilitate the study of mansonellosis in endemic and laboratory settings.

Social effects of rabies infection in male vampire bats (Desmodus rotundus).

Rabies virus (RABV) transmitted by the common vampire bat (Desmodus rotundus) poses a threat to agricultural development and public health throughout the Neotropics. The ecology and evolution of rabies host-pathogen dynamics are influenced by two infection-induced behavioural changes. RABV-infected hosts often exhibit increased aggression which facilitates transmission, and rabies also leads to reduced activity and paralysis prior to death. Although several studies document rabies-induced behavioural changes in rodents and other dead-end hosts, surprisingly few studies have measured these changes in vampire bats, the key natural reservoir throughout Latin America. Taking advantage of an experiment designed to test an oral rabies vaccine in captive male vampire bats, we quantify for the first time, to our knowledge, how rabies affects allogrooming and aggressive behaviours in this species. Compared to non-rabid vampire bats, rabid individuals reduced their allogrooming prior to death, but we did not detect increases in aggression among bats. To put our results in context, we review what is known and what remains unclear about behavioural changes of rabid vampire bats (resumen en español, electronic supplementary material, S1).

Primary infection with dengue or Zika virus does not affect the severity of heterologous secondary infection in macaques.

Zika virus (ZIKV) and dengue virus (DENV) are genetically and antigenically related flaviviruses that now co-circulate in much of the tropical and subtropical world. The rapid emergence of ZIKV in the Americas in 2015 and 2016, and its recent associations with Guillain-Barré syndrome, birth defects, and fetal loss have led to the hypothesis that DENV infection induces cross-reactive antibodies that influence the severity of secondary ZIKV infections. It has also been proposed that pre-existing ZIKV immunity could affect DENV pathogenesis. We examined outcomes of secondary ZIKV infections in three rhesus and fifteen cynomolgus macaques, as well as secondary DENV-2 infections in three additional rhesus macaques up to a year post-primary ZIKV infection. Although cross-binding antibodies were detected prior to secondary infection for all animals and cross-neutralizing antibodies were detected for some animals, previous DENV or ZIKV infection had no apparent effect on the clinical course of heterotypic secondary infections in these animals. All animals had asymptomatic infections and, when compared to controls, did not have significantly perturbed hematological parameters. Rhesus macaques infected with DENV-2 approximately one year after primary ZIKV infection had higher vRNA loads in plasma when compared with serum vRNA loads from ZIKV-naive animals infected with DENV-2, but a differential effect of sample type could not be ruled out. In cynomolgus macaques, the serotype of primary DENV infection did not affect the outcome of secondary ZIKV infection.

Epidemiology and genetic diversity of circulating dengue viruses in Medellin, Colombia: a fever surveillance study.

BACKGROUND: Dengue fever is a major public health problem in Colombia. A fever surveillance study was conducted for evaluation of the clinical, epidemiological, and molecular patterns of dengue, prior to Chikungunya and Zika epidemics. METHODS: In November 2011-February 2014, a passive facility-based surveillance was implemented in Santa Cruz Hospital, Medellin, and enrolled eligible febrile patients between 1 and 65 years-of-age. Acute and convalescent blood samples were collected 10-21 days apart and tested for dengue using IgM/IgG ELISA. RNA was extracted for serotyping using RT-PCR on acute samples and genotyping was performed by sequencing. RESULTS: Among 537 febrile patients enrolled during the study period, 29% (n = 155) were identified to be dengue-positive. Only 7% of dengue cases were hospitalized, but dengue-positive patients were 2.6 times more likely to be hospitalized, compared to non-dengue cases, based on a logistic regression. From those tested with RT-PCR (n = 173), 17 were dengue-confirmed based on PCR and/or virus isolation showing mostly DENV-3 (n = 9) and DENV-4 (n = 7) with 1 DENV-1. Genotyping results showed that: DENV-1 isolate belongs to the genotype V or American/African genotype; DENV-3 isolates belong to genotype III; and DENV-4 isolates belong to the II genotype and specifically to the IIb sub-genotype or linage. CONCLUSIONS: Our surveillance documented considerable dengue burden in Santa Cruz comuna during non-epidemic years, and genetic diversity of circulating DENV isolates, captured prior to Chikungunya epidemic in 2014 and Zika epidemic in 2015. Our study findings underscore the need for continued surveillance and monitoring of dengue and other arboviruses and serve as epidemiological and molecular evidence base for future studies to assess changes in DENV transmission in Medellin, given emerging and re-emerging arboviral diseases in the region.

Immunological Memory to Zika Virus in a University Community in Colombia, South America.

Zika virus appeared in South America in 2015, generating alarm worldwide as it causes microcephaly and autoimmunity. This study aims to determine the serological footprint of the incoming epidemic in a student community and to characterize the memory functional cell response during post convalescence. In a cross-sectional study, Zika-specific IgG using LIA immunoassay was found in 328 university students (CI=95%), while in the second phase, the functional cellular memory response for IFN-γ and IL-2 was quantified using post-stimulus ELISpot with inactivated virus, starting with individuals seropositive for Zika and control individuals (seropositive only for Dengue and seronegative for Zika-Dengue). Depending on the antigen used, memory humoral response (IgG) against Zika Virus was observed in >60% of the population; seropositivity for NS1 was 21.1% higher than E antigen with high intensity. The analysis of cell functionality in 22 individuals seropositive for Zika virus revealed either IFN-γ+ or IL-2+ cells in 86.3% of cases (Th1 profile), presenting multifunctionality in 50% (11 individuals), 64% of which presented> 6 SFC/104 PBMCs (>600 SFC/106 PBMC), reflecting memory circulating cells. A good agreement (Kappa= 0.754) was observed between the coexistence of both cellular and humoral responses but not in their intensity.

Highly efficient maternal-fetal Zika virus transmission in pregnant rhesus macaques.

Infection with Zika virus (ZIKV) is associated with human congenital fetal anomalies. To model fetal outcomes in nonhuman primates, we administered Asian-lineage ZIKV subcutaneously to four pregnant rhesus macaques. While non-pregnant animals in a previous study contemporary with the current report clear viremia within 10-12 days, maternal viremia was prolonged in 3 of 4 pregnancies. Fetal head growth velocity in the last month of gestation determined by ultrasound assessment of head circumference was decreased in comparison with biparietal diameter and femur length within each fetus, both within normal range. ZIKV RNA was detected in tissues from all four fetuses at term cesarean section. In all pregnancies, neutrophilic infiltration was present at the maternal-fetal interface (decidua, placenta, fetal membranes), in various fetal tissues, and in fetal retina, choroid, and optic nerve (first trimester infection only). Consistent vertical transmission in this primate model may provide a platform to assess risk factors and test therapeutic interventions for interruption of fetal infection. The results may also suggest that maternal-fetal ZIKV transmission in human pregnancy may be more frequent than currently appreciated.

Correction to: International prospective observational cohort study of Zika in infants and pregnancy (ZIP study): study protocol.

Following publication of the original article [1], the author mentioned that two additional NIH staff were involved in the development of the protocol who did not receive recognition in the Acknowledgments section in their published article.

International prospective observational cohort study of Zika in infants and pregnancy (ZIP study): study protocol.

BACKGROUND: Until recently, Zika virus (ZIKV) infections were considered mild and self-limiting. Since 2015, they have been associated with an increase in microcephaly and other birth defects in newborns. While this association has been observed in case reports and epidemiological studies, the nature and extent of the relationship between ZIKV and adverse pregnancy and pediatric health outcomes is not well understood. With the unique opportunity to prospectively explore the full spectrum of issues related to ZIKV exposure during pregnancy, we undertook a multi-country, prospective cohort study to evaluate the association between ZIKV and pregnancy, neonatal, and infant outcomes. METHODS: At research sites in ZIKV endemic regions of Brazil (4 sites), Colombia, Guatemala, Nicaragua, Puerto Rico (2 sites), and Peru, up to 10,000 pregnant women will be recruited and consented in the first and early second trimesters of pregnancy and then followed through delivery up to 6 weeks post-partum; their infants will be followed until at least 1 year of age. Pregnant women with symptomatic ZIKV infection confirmed by presence of ZIKV RNA and/or IgM for ZIKV will also be enrolled, regardless of gestational age. Participants will be tested monthly for ZIKV infection; additional demographic, physical, laboratory and environmental data will be collected to assess the potential interaction of these variables with ZIKV infection. Delivery outcomes and detailed infant assessments, including physical and neurological outcomes, will be obtained. DISCUSSION: With the emergence of ZIKV in the Americas and its association with adverse pregnancy outcomes in this region, a much better understanding of the spectrum of clinical outcomes associated with exposure to ZIKV during pregnancy is needed. This cohort study will provide information about maternal, fetal, and infant outcomes related to ZIKV infection, including congenital ZIKV syndrome, and manifestations that are not detectable at birth but may appear during the first year of life. In addition, the flexibility of the study design has provided an opportunity to modify study parameters in real time to provide rigorous research data to answer the most critical questions about the impact of congenital ZIKV exposure. TRIAL REGISTRATION: NCT02856984 . Registered August 5, 2016. Retrospectively registered.

Analyzing the Human Serum Antibody Responses to a Live Attenuated Tetravalent Dengue Vaccine Candidate.

Background: Dengue virus serotypes 1-4 (DENV-1-4) are the most common vector-borne viral pathogens of humans and the etiological agents of dengue fever and dengue hemorrhagic syndrome. A live-attenuated tetravalent dengue vaccine (TDV) developed by Takeda Vaccines has recently progressed to phase 3 safety and efficacy evaluation. Methods: We analyzed the qualitative features of the neutralizing antibody (nAb) response induced in naive and DENV-immune individuals after TDV administration. Using DENV-specific human monoclonal antibodies (mAbs) and recombinant DENV displaying different serotype-specific Ab epitopes, we mapped the specificity of TDV-induced nAbs against DENV-1-3. Results: Nearly all subjects had high levels of DENV-2-specific nAbs directed to epitopes centered on domain III of the envelope protein. In some individuals, the vaccine induced nAbs that tracked with a DENV-1-specific neutralizing epitope centered on domain I of the envelope protein. The vaccine induced binding Abs directed to a DENV-3 type-specific neutralizing epitope, but findings of mapping of DENV-3 type-specific nAbs were inconclusive. Conclusion: Here we provide qualitative measures of the magnitude and epitope specificity of the nAb responses to TDV. This information will be useful for understanding the performance of TDV in clinical trials and for identifying correlates of protective immunity.

Development of Envelope Protein Antigens To Serologically Differentiate Zika Virus Infection from Dengue Virus Infection.

Zika virus (ZIKV) is an emerging flavivirus that can cause birth defects and neurologic complications. Molecular tests are effective for diagnosing acute ZIKV infection, although the majority of infections produce no symptoms at all or present after the narrow window in which molecular diagnostics are dependable. Serology is a reliable method for detecting infections after the viremic period; however, most serological assays have limited specificity due to cross-reactive antibodies elicited by flavivirus infections. Since ZIKV and dengue virus (DENV) widely cocirculate, distinguishing ZIKV infection from DENV infection is particularly important for diagnosing individual cases or for surveillance to coordinate public health responses. Flaviviruses also elicit type-specific antibodies directed to non-cross-reactive epitopes of the infecting virus; such epitopes are attractive targets for the design of antigens for development of serological tests with greater specificity. Guided by comparative epitope modeling of the ZIKV envelope protein, we designed two recombinant antigens displaying unique antigenic regions on domain I (Z-EDI) and domain III (Z-EDIII) of the ZIKV envelope protein. Both the Z-EDI and Z-EDIII antigens consistently detected ZIKV-specific IgG in ZIKV-immune sera but not cross-reactive IgG in DENV-immune sera in late convalescence (>12 weeks postinfection). In contrast, during early convalescence (2 to 12 weeks postinfection), secondary DENV-immune sera and some primary DENV-immune sera cross-reacted with the Z-EDI and Z-EDIII antigens. Analysis of sequential samples from DENV-immune individuals demonstrated that Z-EDIII cross-reactivity peaked in early convalescence and declined steeply over time. The Z-EDIII antigen has much potential as a diagnostic antigen for population-level surveillance and for detecting past infections in patients.

In Vivo Imaging with Bioluminescent Enterovirus 71 Allows for Real-Time Visualization of Tissue Tropism and Viral Spread.

Hand, foot, and mouth disease (HFMD) is a reemerging illness caused by a variety of enteroviruses. The main causative agents are enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and, most recently, coxsackievirus A6 (CVA6). Enterovirus infections can vary from asymptomatic infections to those with a mild fever and blisters on infected individuals' hands, feet, and throats to infections with severe neurological complications. Viral persistence for weeks postinfection (wpi) has also been documented by the demonstration of virus in children's stools. However, little is known about disease progression, viral spread, and tissue tropism of these viruses. These types of studies are limited because many recently developed mouse models mimic the severe neurological complications that occur in a small percentage of enterovirus infections. In the present study, we documented real-time EV71 infection in two different mouse strains by the use of in vivo imaging. Infection of BALB/c mice with a bioluminescent mouse-adapted EV71 construct (mEV71-NLuc) resulted in a lack of clinical signs of disease but in relatively high viral replication, as visualized by luminescence, for 2 wpi. In contrast, mEV71-NLuc infection of AG129 mice (alpha/beta and gamma interferon receptor deficient) showed rapid spread and long-term persistence of the virus in the brain. Interestingly, AG129 mice that survived infection maintained luminescence in the brain for up to 8 wpi. The results we present here will allow future studies on EV71 antiviral drug susceptibility, vaccine efficacy, transmissibility, and pathogenesis. IMPORTANCE We report here that a stable full-length enterovirus 71 (EV71) reporter construct was used to visualize real-time viral spread in AG129 and BALB/c mice. To our knowledge, this is the first report of in vivo imaging of infection with any member of the Picornaviridae family. The nanoluciferase (NLuc) gene, one of the smallest luciferase genes currently available, was shown to be stable in the EV71 genome for eight passages on rhabdomyosarcoma cells. Real-time visualization of EV71 infection in mice identified areas of tropism that would have been missed by traditional methods, including full characterization of EV71 replication in BALB/c mice. Additionally, the bioluminescent construct allowed for increased speed and sensitivity of cell culture assays and will allow future studies involving various degrees of enterovirus infection in mice, not just severe infections. Our data suggest that interferon plays an important role in controlling EV71 infection in the central nervous system of mice.

Risk Factors Associated with Dengue Transmission and Spatial Distribution of High Seroprevalence in Schoolchildren from the Urban Area of Medellin, Colombia.

Dengue fever is an increasing health problem in tropical and subtropical regions. During 2010 in Medellin, the younger population presented a particularly high dengue incidence rate. This study estimated dengue virus (DENV) transmission in schoolchildren (aged 5-19 years) in Medellin from 2010 to 2012. A longitudinal serological survey (IgG) and spatial analysis were conducted to determine the distribution of DENV seroprevalence. A total of 4,385 schoolchildren participated for at least one year. Dengue seroprevalence significantly increased during the studied period (53.8% to 64.6%; p < 0.001). A significantly higher seroconversion rate was observed in 2010-2011 (16.8%) compared to 2011-2012 (7.8%). Multivariate regression analysis showed that the main factor associated with the seroprevalence was the aging. Furthermore, in 2010, patients with high socioeconomic status presented a lower risk. Predominant multitypic and DENV4 monotypic antibody responses were demonstrated. Geostatistical analysis evidenced a temporal clustering distribution of DENV seroprevalence in 2010. Population density and Ae. aegypti House Index were significantly correlated with the observed pattern. This study revealed high DENV transmission in schoolchildren determined as "sentinel population." High DENV risk was found in districts with combined poorly socioeconomic conditions and densest human and mosquito populations. These findings may allow to target population for effective prevention and vaccination campaigns.

Mosaic H5 Hemagglutinin Provides Broad Humoral and Cellular Immune Responses against Influenza Viruses.

UNLABELLED: The most effective way to prevent influenza virus infection is via vaccination. However, the constant mutation of influenza viruses due to antigenic drift and shift compromises vaccine efficacy. This represents a major challenge to the development of a cross-protective vaccine that can protect against circulating viral antigenic diversity. Using the modified vaccinia Ankara (MVA) virus, we had previously generated a recombinant vaccine against highly pathogenic avian influenza virus (H5N1) based on an in silico mosaic approach. This MVA-H5M construct protected mice against multiple clades of H5N1 and H1N1 viruses. We have now further characterized the immune responses using immunodepletion of T cells and passive serum transfer, and these studies indicate that antibodies are the main contributors in homosubtypic protection (H5N1 clades). Compared to a MVA construct expressing hemagglutinin (HA) from influenza virus A/VN/1203/04 (MVA-HA), the MVA-H5M vaccine markedly increased and broadened B cell and T cell responses against H5N1 virus. The MVA-H5M also provided effective protection with no morbidity against H5N1 challenge, whereas MVA-HA-vaccinated mice showed clinical signs and experienced significant weight loss. In addition, MVA-H5M induced CD8(+) T cell responses that play a major role in heterosubtypic protection (H1N1). Finally, expression of the H5M gene as either a DNA vaccine or a subunit protein protected mice against H5N1 challenge, indicating the effectiveness of the mosaic sequence without viral vectors for the development of a universal influenza vaccine. IMPORTANCE: Influenza viruses infect up to one billion people around the globe each year and are responsible for 300,000 to 500,000 deaths annually. Vaccines are still the main intervention to prevent infection, but they fail to provide effective protection against heterologous strains of viruses. We developed broadly reactive H5N1 vaccine based on an in silico mosaic approach and previously demonstrated that modified vaccinia Ankara expressing an H5 mosaic hemagglutinin prevented infection with multiple clades of H5N1 and limited severe disease after H1N1 infection. Further characterization revealed that antibody responses and T cells are main contributors to protection against H5N1 and H1N1 viruses, respectively. The vaccine also broadens both T cell and B cell responses compared to native H5 vaccine from influenza virus A/Vietnam/1203/04. Finally, delivering the H5 mosaic as a DNA vaccine or as a purified protein demonstrated effective protection similar to the viral vector approach.

A Single Mutation in the VP1 of Enterovirus 71 Is Responsible for Increased Virulence and Neurotropism in Adult Interferon-Deficient Mice.

UNLABELLED: Hand, foot, and mouth disease (HFMD) has spread throughout the Asia-Pacific region, affecting millions of young children, who develop symptoms ranging from painful blisters around their mouths and hands to neurological complications. Many members of the genus Enterovirus (family Picornaviridae) cause HFMD. Enterovirus 71 (EV71) is one of the primary causative agents and has been linked to severe disease. Vaccine efficacy and pathogenesis studies for EV71 have been limited because there is a lack of suitable animal models. Previously, we generated a mouse-adapted EV71 (mEV71) capable of infecting 12-week-old interferon receptor-deficient AG129 mice and used the model to evaluate the efficacy of candidate HFMD vaccines. Here, we present data investigating the genetic correlates of EV71 adaptation and characterize the virus's tissue tropism in mice. Using reverse genetics, a VP1 mutation (K244E) was shown to be necessary for mEV71 virulence in adult mice. Another VP1 mutation (H37R) was required for mEV71 recovery on rhabdomyosarcoma (RD) cells. Viral loads determined by real-time reverse transcription (RT)-PCR confirmed the presence of mEV71 in the sera and multiple organs of mice. Histological analysis revealed signs of meningitis and encephalitis, characteristic of severe human disease. The further description of this model has provided insight into EV71 pathogenesis and demonstrates the importance of the VP1 region in facilitating mEV71 adaptation. IMPORTANCE: EV71 is a reemerging pathogen, and little is known about the genetic determinants involved in its pathogenesis. The absence of animal models has contributed to this lack of knowledge. The data presented here improve upon the existing animal models by characterizing a mouse-adapted strain of EV71. We determined that a VP1 mutation (K244E) was needed for EV71 virulence in adult AG129 mice. While this mutation was found previously for EV71 adaptation in 5-day-old BALB/c mice, neurotropic disease did not develop. Using interferon-deficient mice, we raised the age of susceptibility beyond 6 weeks and provided clear evidence that our model mimics severe human infections. The model can be exploited to identify determinants of EV71 virulence and to reveal molecular mechanisms that control the virus-host interaction, especially those associated with neurotropic disease. Furthermore, these data provide useful information regarding the importance of VP1, specifically position 244, in host adaptation and tissue dissemination.

Dissecting the Role of E2 Protein Domains in Alphavirus Pathogenicity.

UNLABELLED: Alphaviruses represent a diverse set of arboviruses, many of which are important pathogens. Chikungunya virus (CHIKV), an arthritis-inducing alphavirus, is the cause of a massive ongoing outbreak in the Caribbean and South America. In contrast to CHIKV, other related alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and Semliki Forest virus (SFV), can cause encephalitic disease. E2, the receptor binding protein, has been implicated as a determinant in cell tropism, host range, pathogenicity, and immunogenicity. Previous reports also have demonstrated that E2 contains residues important for host range expansions and monoclonal antibody binding; however, little is known about what role each protein domain (e.g., A, B, and C) of E2 plays on these factors. Therefore, we constructed chimeric cDNA clones between CHIKV and VEEV or SFV to probe the effect of each domain on pathogenicity in vitro and in vivo. CHIKV chimeras containing each of the domains of the E2 (ΔDomA, ΔDomB, and ΔDomC) from SFV, but not VEEV, were successfully rescued. Interestingly, while all chimeric viruses were attenuated compared to CHIKV in mice, ΔDomB virus showed similar rates of infection and dissemination in Aedes aegypti mosquitoes, suggesting differing roles for the E2 protein in different hosts. In contrast to CHIKV; ΔDomB, and to a lesser extent ΔDomA, caused neuron degeneration and demyelination in mice infected intracranially, suggesting a shift toward a phenotype similar to SFV. Thus, chimeric CHIKV/SFV provide insights on the role the alphavirus E2 protein plays on pathogenesis. IMPORTANCE: Chikungunya virus (CHIKV) has caused large outbreaks of acute and chronic arthritis throughout Africa and Southeast Asia and has now become a massive public health threat in the Americas, causing an estimated 1.2 million human cases in just over a year. No approved vaccines or antivirals exist for human use against CHIKV or any other alphavirus. Despite the threat, little is known about the role the receptor binding protein (E2) plays on disease outcome in an infected host. To study this, our laboratory generated chimeric CHIKV containing corresponding regions of the Semliki Forest virus (SFV) E2 (domains A, B, and C) substituted into the CHIKV genome. Our results demonstrate that each domain of E2 likely plays a critical, but dissimilar role in the viral life cycle. Our experiments show that manipulation of E2 domains can be useful for studies on viral pathogenesis and potentially the production of vaccines and/or antivirals.

CD8+ T-cell Responses in Flavivirus-Naive Individuals Following Immunization with a Live-Attenuated Tetravalent Dengue Vaccine Candidate.

We are developing a live-attenuated tetravalent dengue vaccine (TDV) candidate based on an attenuated dengue 2 virus (TDV-2) and 3 chimeric viruses containing the premembrane and envelope genes of dengue viruses (DENVs) -1, -3, and -4 expressed in the context of the attenuated TDV-2 genome (TDV-1, TDV-3, and TDV-4, respectively). In this study, we analyzed and characterized the CD8(+) T-cell response in flavivirus-naive human volunteers vaccinated with 2 doses of TDV 90 days apart via the subcutaneous or intradermal routes. Using peptide arrays and intracellular cytokine staining, we demonstrated that TDV elicits CD8(+) T cells targeting the nonstructural NS1, NS3, and NS5 proteins of TDV-2. The cells were characterized by the production of interferon-γ, tumor necrosis factor-α, and to a lesser extent interleukin-2. Responses were highest on day 90 after the first dose and were still detectable on 180 days after the second dose. In addition, CD8(+) T cells were multifunctional, producing ≥2 cytokines simultaneously, and cross-reactive to NS proteins of the other 3 DENV serotypes. Overall, these findings describe the capacity of our candidate dengue vaccine to elicit cellular immune responses and support the further evaluation of T-cell responses in samples from future TDV clinical trials.

Safety and Immunogenicity of a Live Attenuated Tetravalent Dengue Vaccine Candidate in Flavivirus-Naive Adults: A Randomized, Double-Blinded Phase 1 Clinical Trial.

BACKGROUND: Dengue viruses (DENVs) infect >300 million people annually, causing 96 million cases of dengue disease and 22 000 deaths [1]. A safe vaccine that protects against DENV disease is a global health priority [2]. METHODS: We enrolled 72 flavivirus-naive healthy adults in a phase 1 double-blinded, randomized, placebo-controlled dose-escalation trial (low and high dose) of a live attenuated recombinant tetravalent dengue vaccine candidate (TDV) given in 2 doses 90 days apart. Volunteers were followed for safety, vaccine component viremia, and development of neutralizing antibodies to the 4 DENV serotypes. RESULTS: The majority of adverse events were mild, with no vaccine-related serious adverse events. Vaccinees reported injection site pain (52% vs 17%) and erythema (73% vs 25%) more frequently than placebo recipients. Low levels of TDV-serotype 2 (TDV-2), TDV-3, and TDV-4 viremia were observed after the first but not second administration of vaccine. Overall seroconversion rates and geometric mean neutralization titers after 2 doses were 84.2% and 54.1, respectively, for DENV serotype 1 (DENV-1); 92.1% and 292.8, respectively, for DENV-2; 86.8% and 32.3, respectively, for DENV-3; and 71.1% and 15.0, respectively, for DENV-4. More than 90.0% of high-dose recipients had trivalent or broader responses. CONCLUSIONS: TDV was generally well tolerated, induced trivalent or broader neutralizing antibodies to DENV in most flavivirus-naive vaccinees, and is undergoing further development. CLINICAL TRIALS REGISTRATION: NCT01110551.