Role of C-terminal domain of Mycobacterium tuberculosis PE6 (Rv0335c) protein in host mitochondrial stress and macrophage apoptosis.

PE/PPE proteins of Mycobacterium tuberculosis (Mtb) target the host organelles to dictate the outcome of infection. This study investigated the significance of PE6/Rv0335c protein's unique C-terminal in causing host mitochondrial perturbations and apoptosis. In-silico analysis revealed that similar to eukaryotic apoptotic Bcl2 proteins, Rv0335c had disordered, hydrophobic C-terminal and two BH3-like motifs in which one was located at C-terminal. Also, Rv0335c's N terminal had mitochondrial targeting sequence. Since, C-terminal of Bcl2 proteins are crucial for mitochondria targeting and apoptosis; it became relevant to evaluate the role of Rv0335c's C-terminal domain in modulating host mitochondrial functions and apoptosis. To confirm this, in-vitro experiments were conducted with Rv0335c whole protein and Rv0335c∆Cterm (C-terminal domain deleted Rv0335c) protein. Rv0335c∆Cterm caused significant reduction in mitochondrial perturbations and Caspase-mediated apoptosis of THP1 macrophages in comparison to Rv0335c. However, the deletion of C-terminal domain didn't affect Rv0335c's ability to localize to mitochondria. Nine Ca2+ binding residues were predicted within Rv0335c and four of them were at the C-terminal. In-vitro studies confirmed that Rv0335c caused significant increase in intracellular calcium influx whereas Rv0335c∆Cterm had insignificant effect on Ca2+ influx. Rv0335c has been reported to be a TLR4 agonist and, we observed a significant reduction in the expression of TLR4-HLA-DR-TNF-α in response to Rv0335c∆Cterm protein also suggesting the role of Rv0335c's C-terminal domain in host-pathogen interaction. These findings indicate the possibility of Rv0335c as a molecular mimic of eukaryotic Bcl2 proteins which equips it to cause host mitochondrial perturbations and apoptosis that may facilitate pathogen persistence.

Late stage specific Rv0109 (PE_PGRS1) protein of Mycobacterium tuberculosis induces mitochondria mediated macrophage apoptosis.

Mitochondria are the powerhouse of the cell and a critical cell signalling hub that decides the fate of the cell. Mycobacterium tuberculosis (Mtb) being a successful pathogen targets and controls the host mitochondria for pathogenesis. Various effector proteins of Mtb are also known to target host mitochondria which include few proteins of a unique Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) family exclusively present in pathogenic mycobacteria, but many of them are still uncharacterized. The present study investigates one such late expressing Rv0109 (PE_PGRS1) protein of Mtb. In-silico analysis predicted the presence of mitochondria targeting signal sequences in Rv0109 and its role in regulation of cysteine type endopeptidase (caspase) activity during apoptosis. Recombinant Rv0109 gets localized to mitochondria of THP1 macrophages as shown by confocal microscopy. Rv0109 was observed to induce mitochondrial stress which resulted in mitochondrial membrane depolarization, upregulation of mitochondrial superoxides and release of Cytochrome-C in the cytoplasm through flow cytometry. Depleted intracellular ATP was observed in THP1 macrophages in response to Rv0109. This mitochondrial stress in response to Rv0109 was observed to culminate in increased expression of pro-apoptotic Bax and Bim factors and caspase activation leading to macrophage apoptosis. Since Rv0109 is a late stage specific protein expressed within granuloma; mitochondria mediated apoptosis induced by Rv0109 may be explored for its role in granuloma maintenance and pathogen persistence.

Efficacy, safety, and lot-to-lot immunogenicity of an inactivated SARS-CoV-2 vaccine (BBV152): interim results of a randomised, double-blind, controlled, phase 3 trial.

BACKGROUND: We report the clinical efficacy against COVID-19 infection of BBV152, a whole virion inactivated SARS-CoV-2 vaccine formulated with a toll-like receptor 7/8 agonist molecule adsorbed to alum (Algel-IMDG) in Indian adults. METHODS: We did a randomised, double-blind, placebo-controlled, multicentre, phase 3 clinical trial in 25 Indian hospitals or medical clinics to evaluate the efficacy, safety, and immunological lot consistency of BBV152. Adults (age ≥18 years) who were healthy or had stable chronic medical conditions (not an immunocompromising condition or requiring treatment with immunosuppressive therapy) were randomised 1:1 with a computer-generated randomisation scheme (stratified for the presence or absence of chronic conditions) to receive two intramuscular doses of vaccine or placebo administered 4 weeks apart. Participants, investigators, study coordinators, study-related personnel, the sponsor, and nurses who administered the vaccines were masked to treatment group allocation; an unmasked contract research organisation and a masked expert adjudication panel assessed outcomes. The primary outcome was the efficacy of the BBV152 vaccine in preventing a first occurrence of laboratory-confirmed (RT-PCR-positive) symptomatic COVID-19 (any severity), occurring at least 14 days after the second dose in the per-protocol population. We also assessed safety and reactogenicity throughout the duration of the study in all participants who had received at least one dose of vaccine or placebo. This report contains interim results (data cutoff May 17, 2021) regarding immunogenicity and safety outcomes (captured on days 0 to 56) and efficacy results with a median of 99 days for the study population. The trial was registered on the Indian Clinical Trials Registry India, CTRI/2020/11/028976, and ClinicalTrials.gov, NCT04641481 (active, not recruiting). FINDINGS: Between Nov 16, 2020, and Jan 7, 2021, we recruited 25 798 participants who were randomly assigned to receive BBV152 or placebo; 24 419 received two doses of BBV152 (n=12 221) or placebo (n=12 198). Efficacy analysis was dependent on having 130 cases of symptomatic COVID-19, which occurred when 16 973 initially seronegative participants had at least 14 days follow-up after the second dose. 24 (0·3%) cases occurred among 8471 vaccine recipients and 106 (1·2%) among 8502 placebo recipients, giving an overall estimated vaccine efficacy of 77·8% (95% CI 65·2-86·4). In the safety population (n=25 753), 5959 adverse events occurred in 3194 participants. BBV152 was well tolerated; the same proportion of participants reported adverse events in the vaccine group (1597 [12·4%] of 12 879) and placebo group (1597 [12·4%] of 12 874), with no clinically significant differences in the distributions of solicited, unsolicited, or serious adverse events between the groups, and no cases of anaphylaxis or vaccine-related deaths. INTERPRETATION: BBV152 was highly efficacious against laboratory-confirmed symptomatic COVID-19 disease in adults. Vaccination was well tolerated with no safety concerns raised in this interim analysis. FUNDING: Bharat Biotech International and Indian Council of Medical Research.

DosR proteins of Mycobacterium tuberculosis upregulate effector T cells and down regulate T regulatory cells in TB patients and their healthy contacts.

It is well established that the current problem of tuberculosis (TB) can be combated by overcoming the drawbacks of the currently available BCG vaccine. This would involve incorporation of antigens that can control TB at all stages including the dormant phase which is generally ignored. Hence, DosR regulon proteins, which are expressed in latent infection, could prove to be very good vaccine candidates as they can possibly target the silent but most predominant form of TB infection. In the present study, the immune response to two DosR proteins Rv2627 and Rv2628 has been studied in PBMCs derived from normal individuals, TB patients and healthy contacts of TB patients. It was found that these antigens were capable of stimulating a strong IFN-γ+ T cell response along with accentuation of memory T cells and other protective cytokines such as IL-2 and IL-17. At the same time these proteins decreased the frequencies of immune-suppressor regulatory T cells in in vitro stimulation of PBMC from both patients and their contacts. Considering all these facts together, we suggest Rv2627 and Rv2628 to be one of the extremely promising candidates for incorporation into a post exposure subunit vaccine against TB.

Mycobacterium tuberculosis dormancy regulon proteins Rv2627c and Rv2628 as Toll like receptor agonist and as potential adjuvant.

During latency, DosR proteins of Mycobacterium tuberculosis (M.tb) get activated and help the bacterium to remain dormant. We have shown earlier that 2 such proteins Rv2627c and Rv2628 are immunogenic and induce a TH1 kind of immune response. In this study, through in-vitro experiments we have confirmed that Rv2627c and Rv2628 proteins act as protein Toll-Like Receptor (TLR) agonist-adjuvant. Rv2627c and Rv2628 stimulated THP-1 macrophages showed an increased expression of TLR2, TLR4 and co-stimulatory molecules CD40, CD80, CD86 and antigen presenting molecule HLA-DR. Further studies also found enhanced expression of downstream signaling molecules of TLR activation like MyD88, NF-κB-p65 and pro-inflammatory cytokines. Inhibition studies using TLR blocking antibodies decreased the expression of co-stimulatory molecules, MyD88, NF-κB-p65, and pro-inflammatory cytokines. Rv2627c and Rv2628 stimulation of HEK-TLR2 reporter cell line confirmed the interaction of these proteins with TLR2. Moreover, molecular docking and simulations of Rv2627c and Rv2628 proteins with TLR2 and TLR4 showed stable interactions. The adjuvant activity of Rv2628 was further validated by a protein adjuvanted with pre-clinically validated peptides as multi-epitope vaccine construct which showed good binding with TLR2 and TLR4 and activate dendritic cells and induce sustained pro-inflammatory cytokine response by C-ImmSim analysis. We propose that our vaccine construct will produce a better immune response than BCG and can be taken up as a post-exposure therapeutic subunit vaccine along with standard TB therapy. We also anticipate that our construct can be taken up as a protein adjuvant with other vaccine candidates as these can activate macrophages through TLR signaling.

Prediction and identification of T cell epitopes of COVID-19 with balanced cytokine response for the development of peptide based vaccines.

Recent outbreak of 2019 novel Corona virus poses serious challenge for the global health system. In lieu of paucity of experimental data, tools and the very basic understanding of host immune responses against SARS-CoV-2, well thought effective measures are needed to control COVID-19 pandemic. We have identified specific overlapping antigenic peptide epitopes (OAPE) within the 4 structural proteins of SARS-CoV-2 predictive of triggering robust CD4 and CD8 T cell responses in host using bio-informatics tools (NetMHC4.0, IEDB, and Vaxijen2.0). We speculate an early release of pro-inflammatory cytokines for protection and later release of anti-inflammatory cytokines for prevention of immunopathology in designing a vaccine for Covid-19. Therefore, the selected immunogenic OAPE were subjected to in silico tools (IL-6-Pred, IFNepitope and PIP-EL) for analyzing their pro-inflammatory response. The OAPEs found to be pro-inflammatory in nature were further subjected to prediction servers (IL-4-Pred, IL-10-Pred, Pre-AIP) to characterize them as inducers of anti-inflammatory response as well. We finally filtered out 12 OAPE which had affinity for both CD4 and CD8 T cells as well as were inducers of pro-inflammatory and anti-inflammatory cytokines. On confirmation of OAPE binding affinity for respective T cell specific MHC allele using docking studies (pepATTRACT, Hex8.0 and Discovery studio) they were found to be have more immunogenic potential than the 3 negative control peptides (NCPs) included in the study. Additionally, we constructed CTxB-adjuvanated multi-epitopic vaccine inclusive of the 12 OAPEs which was non-toxic, non-allergenic and capable of inducing both pro-inflammatory and anti-inflammatory cytokines. A successful in silico cloning and docking of modeled subunit vaccine construct with toll like receptor-2 (TLR-2) confirmed the high efficacy of our multi-epitopic vaccine which can through a balanced interplay of cytokines help in creating a steady-state immune equilibrium. In silico immune simulation studies with the vaccine using C-ImmSim server also showed higher percentage of T cells along with production of pro-inflammatory as well as some anti-inflammatory cytokines. Experimental validation of this prediction based study on Peripheral Blood Mononuclear Cells (PBMCs) of un-infected individuals, patients and recovered individuals will facilitate production of high priority effective SARS -CoV-2 vaccine candidate. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40203-021-00098-7.

Immunogenicity of late stage specific peptide antigens of Mycobacterium tuberculosis.

Global burden of latent TB infection comprises one-third of the world population. Identifying potential Mycobacterium tuberculosis (Mtb) latency associated antigens that can generate protective immunity against the pathogen is crucial for designing an effective TB vaccine. Usually the immune system responds to a small number of amino acids as MHC Class I or Class II peptides. The precision to trigger epitope specific protective T-cell immune response could therefore be achieved with synthetic peptide-based subunit vaccine. In the present study we have considered an immunoinformatic approach using available softwares (ProPred, IEDB, NETMHC, BIMAS, Vaxijen2.0) and docking and visualizing softwares (CABSDOCK, HEX, Pymol, Discovery Studio) to select 10 peptides as latency antigens from 4 proteins (Rv2626, Rv2627, Rv2628, and Rv2032) of DosR regulon of Mtb. As Intracellular IFN-γ secreted by T cells is the most essential cytokine in Th1 mediated protective immunity, these peptides were verified as potential immunogenic epitopes in Peripheral Blood Mononuclear Cells (PBMCs) of 10 healthy contacts of TB patients (HTB) and 10 Category I Pulmonary TB patients (PTB).The antigen-specific CD4 and CD8 T cells expressing intracellular IFN-γ were analyzed using monoclonal antibodies in all subjects by multi-parameter flow cytometry. Both, PTB and HTB individuals responded to DosR peptides by showing increased frequency of IFN-γ+CD4 and IFN-γ+CD8 T cells. The T-cell responses were significantly higher in PTB patients in comparision to the HTB individuals. Additionally, our synthetic peptides and pools showed higher frequencies of IFN-γ+CD4 and IFN-γ+CD8 T cells than the peptides of Ag85B. This pilot study can be taken up further in larger sample size which may support the untapped opportunity of designing Mtb DosR inclusive peptide based post-exposure subunit vaccine.

Increasing rates of infants hospitalized with pertussis.

INTRODUCTION: Four infant deaths in one year due to pertussis at our hospital prompted a retrospective chart review to determine the trends in pertussis hospitalizations in children < one year of age over the past 14 years. METHODS: All medical records from July 1988-June 2002 with pertussis-specific ICD-9 codes were reviewed. Subjects were included in the analysis if they were < one year of age, had positive pertussis tests, an epidemiological link to a laboratory-confirmed case; or if they had cough > two weeks with paroxysms, inspiratory whoops or post-tussive vomiting. Demographic, clinical, laboratory, and hospital course data were collected. RESULTS: Of the 85 patients identified, 69 subjects met the inclusion criteria. Cases increased over the years with two cases detected from 1988-92, 22 from 1993-97, and 45 from 1998-2002 (p < 0.001). Mean age at admission was 71 days; 94 percent were < six mos. compared to children between six mos.-one yr. (p < 0.001). Mean birth weight was 3027g, with 17 percent preterm. Most subjects (77 percent) had no pertussis immunizations. Presenting symptoms were paroxysmal cough 85 percent, cyanosis 68 percent, and apnea 52 percent. Oxygen was required in 35 percent, mechanical ventilation in 12 percent, and extracorporeal membrane oxygenation (ECMO) in four percent because of uncontrolled pulmonary hypertension. All three subjects on ECMO died. Mean lymphocyte count was 46.3 thou/uL in survivors and 61.1 thou/uL in those who died (p = 0.001). CONCLUSIONS: The number of patients hospitalized with pertussis has significantly increased. Mortality was seen in infants < 3 mos. with pulmonary hypertension, pneumonia, and elevated lymphocyte counts. These data support the need for new immunization strategies to prevent pertussis disease in infants too young to have been immunized.