Continual circulation of ECSA genotype and identification of a novel mutation I317V in the E1 gene of Chikungunya viral strains in southern India during 2015-2016.

Chikungunya, a mosquito-borne disease caused by Chikungunya virus (CHIKV), continues to be a significant public health problem in India. In 2016, 56 000 cases were reported from India, the largest number since the reemergence of CHIKV in this region in 2006. In the present study, using molecular and phylogenetic methods, the circulating strains from southern India during 2015-2016 were characterized in the context of circulating Asian strains. Partial envelope gene (E1) sequencing was performed on 20 serum samples positive for CHIKV by a reverse transcription-polymerase chain reaction. Phylogenetic analysis showed that all the sequences in this study belonged to the East Central South African (ECSA) genotype and clustered together with other strains from India. Bayesian phylogenetic analysis revealed that the sequences from the study grouped into two different subclades. The estimate of divergence times suggests that subclades of the ECSA genotype, share a common ancestor approximately 4 to 12 years ago. Six nonsynonymous mutations-K211E, M269V, D284E, V322A, I317V and V220I were noted in E1. In conclusion, this study revealed the cocirculation of distinct subclades within the ECSA genotype of CHIKV in South India during 2015-2016. The I317V mutation in E1 has only been described in recent CHIKV strains from north-central India and Bangladesh. This study highlights the need for continued molecular surveillance to identify the emergence of novel strains and unique mutations in CHIKV with epidemic potential.

Alterations in natural killer and dendritic cell subsets in individuals with HIV-associated neurotuberculosis.

One of the commonest HIV-associated opportunistic infections of the central nervous system is neurotuberculosis. Interaction between HIV, Mycobacterium tuberculosis and host immune system in co-infected individuals may result in altered frequencies of immune cells, thereby modulating dissemination and disease progression. We examined the frequencies of natural killer (NK) cell and dendritic cell (DC) subsets in HIV infected individuals with neurotuberculosis (HIVNTB) as compared to individuals with HIV associated systemic TB (HIVSTB), asymptomatic HIV, non-HIV NTB, non-HIV STB, and healthy controls. Peripheral blood mononuclear cells (PBMC) were stained with fluorochrome-conjugated monoclonal antibodies- Lineage cocktail (containing CD3, CD14, CD19, and CD20), HLA-DR, CD16, CD56, CD11c, and CD123, fixed with 2% paraformaldehyde and analyzed on the flow cytometer. The pDCs were significantly reduced in all HIV infected groups, with a marked reduction in HIVNTB cases as compared to healthy controls. While the CD56- CD16bt NK cell subset displayed a significant increase in frequency in all three HIV infected groups compared the three HIV negative groups, the CD56dim CD16bt subset was significantly lower in frequency in the HIVNTB compared to healthy controls. The decreased frequencies of plasmacytoid DCs and cytotoxic NK cells, which are crucial for innate immune defence against HIV, may result in ineffective virus control and lead to an exacerbated course of disease in HIVNTB individuals.

CD8+ T-cells target the Crimean-Congo haemorrhagic fever virus Gc protein to control the infection in wild-type mice.

BACKGROUND: Crimean-Congo haemorrhagic fever (CCHF) is a serious viral hemorrhagic fever caused by the CCHF virus (CCHFV). Spread by the bites of infected ticks or handling of viremic livestock, human disease is characterized by a non-specific febrile illness that can rapidly progress to fatal hemorrhagic disease. No vaccines or antivirals are available. Case fatality rates can vary but can be higher than 30%, although sub-clinical infections are often unrecognized and unreported. Yet, while most humans infected with CCHFV will survive the infection, often with little-to-no symptoms, the host responses that control the infection are unknown. METHODS: Here we investigated the role of cellular immunity in control of CCHFV infection in an immunocompetent mouse model. FINDINGS: We found that CD8+ T-cells are crucial for efficient control of the acute infection and rapidly acquired CCHFV-specific antiviral effector functions such as production of antiviral cytokines and degranulating in response to CCHFV peptides. We further identified the minimal CD8+ T-cell epitopes in the viral Gc proteins and that infection of mice lacking IFNγ resulted in worsened disease and higher viral loads. INTERPRETATION: Together our data suggest that CD8+ T-cells are important for control of acute CCHFV infection likely through production of antiviral cytokines. FUNDING: This work was supported by the Intramural Research Program of the NIH.

Favipiravir and Ribavirin protect immunocompetent mice from lethal CCHFV infection.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes Crimean-Congo hemorrhagic fever (CCHF) in humans with high morbidity and mortality. Currently, there is neither an approved antiviral drug nor a vaccine against CCHFV. In this study, we describe a lethal model of CCHFV infection using a mouse-adapted strain of CCHFV (MA-CCHFV) in adult wild-type male mice. Infected mice developed high viral loads, tissue pathology, and inflammatory immune responses before ultimately succumbing to the infection. We used the model to evaluate the protective efficacy of nucleoside analogs monulpiravir, favipiravir, ribavirin, the antibiotic tigecycline and the corticosteroids dexamethasone and methylprednisolone against lethal CCHFV infection. Tigecycline, monulpiravir and the corticosteroids failed to protect mice from lethal MA-CCHFV infection. In contrast, favipiravir and ribavirin protected animals from clinical disease and death even when treatment was delayed. Despite demonstrating uniform protection, CCHFV RNA persisted in survivors treated with favipiravir and ribavirin. Nevertheless, the study demonstrated the anti-CCHFV efficacy of favipiravir and ribavirin in a model with intact innate immunity and establishes this model for continued development of CCHFV countermeasures.

CD47 expression attenuates Ebola virus-induced immunopathology in mice.

It has been shown that a very early cell-intrinsic response to infection is the upregulation of CD47 cell surface expression, a molecule known for delivering a "don't eat me signal" that inhibits macrophage-mediated phagocytosis and antigen presentation. Thus, blockade of CD47 signaling during lymphocytic choriomenigitis virus infections of mice has been shown to enhance the kinetics and potency of immune responses, thereby producing faster recovery. It seems counterintuitive that one of the earliest responses to infection would be immunoinhibitory, but it has been hypothesized that CD47 induction acts as an innate immune system checkpoint to prevent immune overactivation and immunopathogenic responses during certain infections. In the current study we examined the effect of CD47 blockade on lethal Ebola virus infection of mice. At 6 days post-infection, CD47 blockade was associated with significantly increased activation of B cells along with increases in recently cytolytic CD8+ T cells. However, the anti-CD47-treated mice exhibited increased weight loss, higher virus titers, and succumbed more rapidly. The anti-CD47-treated mice also had increased inflammatory cytokines in the plasma indicative of a "cytokine storm". Thus, in the context of this rapid hemorrhagic disease, CD47 blockade indeed exacerbated immunopathology and disease severity.

Replicating RNA vaccination elicits an unexpected immune response that efficiently protects mice against lethal Crimean-Congo hemorrhagic fever virus challenge.

BACKGROUND: Crimean-Congo hemorrhagic fever virus is the cause of a severe hemorrhagic fever with cases reported throughout a wide-geographic region. Spread by the bite of infected ticks, contact with infected livestock or in the health care setting, disease begins as a non-specific febrile illness that can rapidly progress to hemorrhagic manifestations. Currently, there are no approved vaccines and antivirals such as ribavirin have unclear efficacy. Thus treatment is mostly limited to supportive care. METHODS: In this report we evaluated an alphavirus-based replicon RNA vaccine expressing either the CCHFV nucleoprotein or glycoprotein precursor in a stringent, heterologous lethal challenge mouse model. FINDINGS: Vaccination with the RNA expressing the nucleoprotein alone could confer complete protection against clinical disease, but vaccination with a combination of both the nucleoprotein and glycoprotein precursor afforded robust protection against disease and viral replication. Protection from lethal challenge required as little as a single immunization with 100ng of RNA. Unexpectedly, analysis of the immune responses elicited by the vaccine components showed that vaccination resulted in antibodies against the internal viral nucleoprotein and cellular immunity against the virion-exposed glycoproteins. INTERPRETATION: Cumulatively this vaccine conferred robust protection against Crimean-Congo hemorrhagic fever virus and supports continued development of this vaccine candidate. FUNDING: This research was supported by the Intramural Research Program of the NIAID/NIH and HDT Bio.

CCL2: a Chemokine Potentially Promoting Early Seeding of the Latent HIV Reservoir.

HIV infects long-lived CD4 memory T cells, establishing a latent viral reservoir that necessitates lifelong antiretroviral therapy (ART). How this reservoir is formed so quickly after infection remains unclear. We now show the innate inflammatory response to HIV infection results in CCL2 chemokine release, leading to recruitment of cells expressing the CCR2 receptor, including a subset of central memory CD4 T cells. Supporting a role for the CCL2/CCR2 axis in rapid reservoir formation, we find (i) treatment of humanized mice with anti-CCL2 antibodies during early HIV infection decreases reservoir seeding and preserves CCR2/5+ cells and (ii) CCR2/5+ cells from the blood of HIV-infected individuals on long-term ART contain significantly more integrated provirus than CCR2/5-negative memory or naive cells. Together, these studies support a model where the host's innate inflammatory response to HIV infection, including CCL2 production, leads to the recruitment of CCR2/5+ central memory CD4 T cells to zones of virus-associated inflammation, likely contributing to rapid formation of the latent HIV reservoir. IMPORTANCE There are currently over 35 million people living with HIV worldwide, and we still have no vaccine or scalable cure. One of the difficulties with HIV is its ability to rapidly establish a viral reservoir in lymphoid tissues that allows it to elude antivirals and the immune system. Thus, it is important to understand how HIV accomplishes this so we can develop preventive strategies. Our current results show that an early inflammatory response to HIV infection includes production of the chemokine CCL2, which recruits a unique subset of CCR2/5+ CD4+ T cells that become infected and form a significant reservoir for latent infection. Furthermore, we show that blockade of CCL2 in humanized mice significantly reduces persistent HIV infection. This information is relevant to the development of therapeutics to prevent and/or treat chronic HIV infections.

Replicating RNA platform enables rapid response to the SARS-CoV-2 Omicron variant and elicits enhanced protection in naïve hamsters compared to ancestral vaccine.

BACKGROUND: In late 2021, the SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VoC) was reported with many mutations in the viral spike protein that were predicted to enhance transmissibility and allow viral escape of neutralizing antibodies. Within weeks of the first report of B.1.1.529, this VoC has rapidly spread throughout the world, replacing previously circulating strains of SARS-CoV-2 and leading to a resurgence in COVID-19 cases even in populations with high levels of vaccine- and infection-induced immunity. Studies have shown that B.1.1.529 is less sensitive to protective antibody conferred by previous infections and vaccines developed against earlier lineages of SARS-CoV-2. The ability of B.1.1.529 to spread even among vaccinated populations has led to a global public health demand for updated vaccines that can confer protection against B.1.1.529. METHODS: We rapidly developed a replicating RNA vaccine expressing the B.1.1.529 spike and evaluated immunogenicity in mice and hamsters. We also challenged hamsters with B.1.1.529 and evaluated whether vaccination could protect against viral shedding and replication within respiratory tissue. FINDINGS: We found that mice previously immunized with A.1-specific vaccines failed to elevate neutralizing antibody titers against B.1.1.529 following B.1.1.529-targeted boosting, suggesting pre-existing immunity may impact the efficacy of B.1.1.529-targeted boosters. Furthermore, we found that our B.1.1.529-targeted vaccine provides superior protection compared to the ancestral A.1-targeted vaccine in hamsters challenged with the B.1.1.529 VoC after a single dose of each vaccine. INTERPRETATION: Our data suggest that B.1.1.529-targeted vaccines may provide superior protection against B.1.1.529 but pre-existing immunity and timing of boosting may need to be considered for optimum protection. FUNDING: This research was supported in part by the Intramural Research Program, NIAID/NIH, Washington Research Foundation and by grants 27220140006C (JHE), AI100625, AI151698, and AI145296 (MG).

Recovery from Acute SARS-CoV-2 Infection and Development of Anamnestic Immune Responses in T Cell-Depleted Rhesus Macaques.

Severe coronavirus disease 2019 (COVID-19) has been associated with T cell lymphopenia, but no causal effect of T cell deficiency on disease severity has been established. To investigate the specific role of T cells in recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, we studied rhesus macaques that were depleted of either CD4+, CD8+, or both T cell subsets prior to infection. Peak virus loads were similar in all groups, but the resolution of virus in the T cell-depleted animals was slightly delayed compared to that in controls. The T cell-depleted groups developed virus-neutralizing antibody responses and class switched to IgG. When reinfected 6 weeks later, the T cell-depleted animals showed anamnestic immune responses characterized by rapid induction of high-titer virus-neutralizing antibodies, faster control of virus loads, and reduced clinical signs. These results indicate that while T cells play a role in the recovery of rhesus macaques from acute SARS-CoV-2 infections, their depletion does not induce severe disease, and T cells do not account for the natural resistance of rhesus macaques to severe COVID-19. Neither primed CD4+ nor CD8+ T cells appeared critical for immunoglobulin class switching, the development of immunological memory, or protection from a second infection. IMPORTANCE Patients with severe COVID-19 often have decreased numbers of T cells, a cell type important in fighting most viral infections. However, it is not known whether the loss of T cells contributes to severe COVID-19 or is a consequence of it. We studied rhesus macaques, which develop only mild COVID-19, similar to most humans. Experimental depletion of T cells slightly prolonged their clearance of virus, but there was no increase in disease severity. Furthermore, they were able to develop protection from a second infection and produced antibodies capable of neutralizing the virus. They also developed immunological memory, which allows a much stronger and more rapid response upon a second infection. These results suggest that T cells are not critical for recovery from acute SARS-CoV-2 infections in this model and point toward B cell responses and antibodies as the essential mediators of protection from re-exposure.

Recovery from acute SARS-CoV-2 infection and development of anamnestic immune responses in T cell-depleted rhesus macaques.

Severe COVID-19 has been associated with T cell lymphopenia 1,2, but no causal effect of T cell deficiency on disease severity has been established. To investigate the specific role of T cells in recovery from SARS-CoV-2 infections we studied rhesus macaques that were depleted of either CD4+, CD8+ or both T cell subsets prior to infection. Peak virus loads were similar in all groups, but the resolution of virus in the T cell-depleted animals was slightly delayed compared to controls. The T cell-depleted groups developed virus-neutralizing antibody responses and also class-switched to IgG. When re-infected six weeks later, the T cell-depleted animals showed anamnestic immune responses characterized by rapid induction of high-titer virus-neutralizing antibodies, faster control of virus loads and reduced clinical signs. These results indicate that while T cells play a role in the recovery of rhesus macaques from acute SARS-CoV-2 infections, their depletion does not induce severe disease, and T cells do not account for the natural resistance of rhesus macaques to severe COVID-19. Neither primed CD4+ or CD8+ T cells appeared critical for immunoglobulin class switching, the development of immunological memory or protection from a second infection.

Alteration of T Cell Phenotypes in HIV-Neurotuberculosis Coinfection.

BACKGROUND: Neurotuberculosis is one of the commonest HIV associated opportunistic infections of the central nervous system in India. HIV-TB coinfection may lead to altered frequencies of T cells, thereby influencing the course and progression of the disease. METHODS: We examined the frequencies of T cell subsets in HIV infected individuals with neurotuberculosis (HIV+nTB+) as compared to individuals with HIV associated systemic TB (HIV+sTB+), asymptomatic HIV (HIV+TB-), non-HIV neuro TB (HIV-nTB+), non-HIV systemic TB (HIV-sTB+), and healthy controls (HIV-TB-). Activation and senescence profiles of CD4 and CD8 T cells and memory subsets in peripheral blood mononuclear cells were studied by flow cytometry. RESULTS: The significant observations among the T cell subsets in HIV+nTB+ were: (1) Naïve T cells: decreased CD4 T cells compared to HIV-sTB+ (P = 0.005); decreased CD8 T cells compared to HIV-nTB+ and HIV-TB- (P ≤ 0.007), (2) Memory T cells: expanded CD4 TEMRA cells compared to HIV-nTB+, HIV-sTB+, and HIV-TB- (P ≤ 0.003); expanded CD8 TEMRA cells compared to HIV-nTB+ and HIV-TB- (P ≤ 0.005), (3) Activated T cells: higher CD4 T cells compared to HIV-nTB+, HIV-sTB+, and HIV-TB- (P ≤ 0.004); higher CD8 T cells compared to HIV + TB-, HIV-nTB+, HIV-sTB+, and HIV-TB- (P ≤ 0.001), and (4) Senescent T cells: increased CD8 T cells compared to HIV-nTB+ and HIV-TB- groups (P = 0.000). CONCLUSIONS: Increased activation compared to HIV+TB-, HIV-nTB+, HIV-sTB+, and HIV-TB- groups and increased senescence compared to HIV-nTB+ and HIV-TB- groups were observed in CD8 T cells in HIV+nTB+, suggesting that the frequencies of these T cell subsets are altered to a greater extent in these individuals. © 2018 International Clinical Cytometry Society.

Pattern recognition receptor mRNA expression and cytokine and granzyme levels in HIV infected individuals with neurotuberculosis.

Neurotuberculosis is one of the commonest HIV-associated opportunistic infections (OI) of the CNS. Cross-talk between HIV, Mycobacterium tuberculosis and host immune responses may alter expression of pattern recognition receptors (PRRs), thereby affecting cytokine profiles and functional responses. We examined PRR mRNA expression and cytokine and granzyme levels in HIV infected individuals with neurotuberculosis and found significant downregulation of TLR9 and increased MDA5 expression compared to healthy subjects. Significantly higher Granzyme A and IFN-γ levels were also observed in the CSF of this group compared to CSF from non-infectious controls. These alterations may lead to inappropriate recruitment of immune cells to the CNS, leading to disease severity.