Manish Bansal

Zoonotic cases of camelpox infection in India

This study reports the first conclusive evidence of zoonotic camelpox virus (CMLV) infection in humans associated with outbreaks in dromedarian camels (Camelus dromedaries) in northwest region of India during 2009. CMLV infection is usually restricted to camels and causes localised skin lesions but occasionally leads to generalised form of disease. However, the present outbreak involved camel handlers and attendants with clinical manifestations such as papules, vesicles, ulceration and finally scabs over fingers and hands. In camels, the pock-like lesions were distributed over the hairless parts of the body. On the basis of clinical and epidemiological features coupled with serological tests and molecular characterization of the causative agent, CMLV zoonosis was confirmed in three human cases. Clinical samples such as skin scabs/swabs and blood collected from affected animals and humans were analysed initially, for the presence of CMLV-specific antigen and antibodies by counter immunoelectrophoresis (CIE); serum neutralization test (SNT); plaque-reduction neutralization test (PRNT) and indirect immunoperoxidase test which was later confirmed by amplification of CMLV-specific ankyrin repeat protein (C18L) gene. Virus isolation was successful only from samples collected from camels. Further, sequence analyses based on three full-length envelope protein genes (A27L, H3L and D8L) revealed 95.2-99.8% and 93.1-99.3% homology with other Orthopoxviruses at nucleotide and amino acid levels, respectively. Phylogram of the three genes revealed a close relationship of CMLV with Variola virus (VARV). Considering the emerging and re-emerging nature of the virus, its genetic relatedness to VARV, zoonotic potential and productivity losses in camels; the control measures are imperative in curtailing economic and public health impact of the disease. This is the first instance of laboratory confirmed camelpox zoonosis in India.

Conformational Epitope-Specific Broadly Neutralizing Plasma Antibodies Obtained from an HIV-1 Clade C-Infected Elite Neutralizer Mediate Autologous Virus Escape through Mutations in the V1 Loop

ABSTRACT Broadly neutralizing antibodies isolated from infected patients who are elite neutralizers have identified targets on HIV-1 envelope (Env) glycoprotein that are vulnerable to antibody neutralization; however, it is not known whether infection established by the majority of the circulating clade C strains in Indian patients elicit neutralizing antibody responses against any of the known targets. In the present study, we examined the specificity of a broad and potent cross-neutralizing plasma obtained from an Indian elite neutralizer infected with HIV-1 clade C. This plasma neutralized 53/57 (93%) HIV pseudoviruses prepared with Env from distinct HIV clades of different geographical origins. Mapping studies using gp120 core protein, single-residue knockout mutants, and chimeric viruses revealed that G37080 broadly cross-neutralizing (BCN) plasma lacks specificities to the CD4 binding site, gp41 membrane-proximal external region, N160 and N332 glycans, and R166 and K169 in the V1-V3 region and are known predominant targets for BCN antibodies. Depletion of G37080 plasma with soluble trimeric BG505-SOSIP.664 Env (but with neither monomeric gp120 nor clade C membrane-proximal external region peptides) resulted in significant reduction of virus neutralization, suggesting that G37080 BCN antibodies mainly target epitopes on cleaved trimeric Env. Further examination of autologous circulating Envs revealed the association of mutation of residues in the V1 loop that contributed to neutralization resistance. In summary, we report the identification of plasma antibodies from a clade C-infected elite neutralizer that mediate neutralization breadth via epitopes on trimeric gp120 not yet reported and confer autologous neutralization escape via mutation of residues in the V1 loop. IMPORTANCE A preventive vaccine to protect against HIV-1 is urgently needed. HIV-1 envelope glycoproteins are targets of neutralizing antibodies and represent a key component for immunogen design. The mapping of epitopes on viral envelopes vulnerable to immune evasion will aid in defining targets of vaccine immunogens. We identified novel conformational epitopes on the viral envelope targeted by broadly cross-neutralizing antibodies elicited in natural infection in an elite neutralizer infected with HIV-1 clade C. Our data extend our knowledge on neutralizing epitopes associated with virus escape and potentially contribute to immunogen design and antibody-based prophylactic therapy.

Laboratory-acquired Buffalopox Virus Infection, India

To the Editor: In India, buffalopox virus (BPXV), a variant of vaccinia virus, is associated with severe disease outbreaks among buffaloes (1,2), cattle (3), and humans in contact with these animals (1,4). Most human BPXV infections occur in animal attendants and milkers (1,4). A similar type of vaccinia virus infection has also been reported from rural areas in Brazil (5). We report a case of laboratory-acquired infection with BPXV in a researcher in India. Clinical signs, symptoms, diagnosis, and management of this case highlight the need for observance and enforcement of strict biosafety measures within the laboratory. n nA 28-year-old man (researcher) who was freeze-drying BPXV isolates in a laboratory in Hisar, India sustained a cut on his right palm through nitrile gloves by accidental piercing of shrapnel from a broken ampule. The virus being freeze-dried (105.5 50% tissue culture infectious doses/mL) was isolated from a buffalo in Jalgaon, India, in 2010. The injured site on the palm was immediately cleaned with 70% ethanol and treated with povidone–iodine solution. No untoward reaction was observed ≤2 days postinjury. Erythema appeared at the injury site on postinjury day 3. Subsequently, a small vesicle developed on postinjury day 5 (Figure, panel A). This vesicle progressed into a pustule with a central area of necrosis by postinjury day 7 (Figure, panel B). On postinjury day 9, symptoms worsened (onset of high fever and general malaise and pain at the affected site), and the researcher sought medical care. n n n nFigure n nProgression of lesion on palm of researcher caused by buffalopox virus infection, India. A) Small vesicle on postinjury day 5. B) Pustule with a central area of necrosis on postinjury day 7. C) Pustule with edema on postinjury day 9. D) Increase in edema ... n n n nPhysical examination showed high fever (104°F), unilateral axillary lymphadenopathy, and edema of the palm (Figure, panel C). Amoxicillin (500 mg, 2×/d), cephalexin (500 mg, 2×/d), and analgesic/antipyretic (paracetamol, 500 mg, 2×/d) were prescribed to control secondary complications caused by bacterial infection and pain. n nThe next day, the entire palm became cyanotic and edema increased (Figure, panel D). The researcher was then referred to a specialty hospital where the lesion was surgically excised on postinjury day 11 (Figure, panel E) under axial block anesthesia. Blood, necrotic tissue, pustular material, and swab specimens were obtained for laboratory examination. Postsurgery treatment included cleaning of the surgical site on alternate days and oral medication (amoxicillin/clavulanic acid, 625 mg; ibuprofen, 400 mg; paracetamol, 325 mg; and rabeprazole, 20 mg) for 5 days. n nOn postinjury day 19, the surgeon advised the patient to take cefuroxime (500 mg/d for 5 days) and use a topical ointment containing mupirocin to prevent a delay in healing (Figure, panel F). The lesion healed slowly, and by postinjury day 30, thickening and blackening of the skin was observed (Figure, panel G) that extended to a wider area by postinjury day 38, and the skin started to peel off by postinjury day 50. The entire skin of the palm sloughed off with complete healing by postinjury day 85, leaving a 20-mm blackened eschar over the area (Figure, panel H). n nClinical samples were subjected to laboratory examination. Virus was isolated from tissue samples in a Vero cell line during the first passage. BPXV infection was confirmed by PCR amplification of orthopoxvirus-specific A type inclusion gene (552 bp) and a BPXV-specific C18L gene (368 bp) from tissue material and the laboratory-isolated virus (BPXV/Human/Lab/11), according to procedures described by Singh et al. (6). Sequences of these 2 genes were submitted to GenBank under accession nos. {type:entrez-nucleotide,attrs:{text:JN653284.1,term_id:346682495,term_text:JN653284.1}}JN653284.1 and {type:entrez-nucleotide,attrs:{text:JN653278.1,term_id:346682489,term_text:JN653278.1}}JN653278.1, respectively. Phylogenetic analysis showed 95%–100% nt similarity of the laboratory isolate (BPXV/Human/Lab/11) with other BPXVs from India (Technical Appendix Figure). Antibodies against BPXV were detected in patient serum samples by using an indirect immunoperoxidase test and a 50% plaque-reduction neutralization test according to methods reported by Bera et al. (7). Serum of the infected patient showed 50% plaque-reduction neutralization test titers of 256 and 512 on postinjury days 11 and 28, respectively. These findings confirmed BPXV infection because the patient had not been vaccinated against smallpox. n nFreeze-drying of the glass ampule to −80°C caused a hairline crack in the glass. The ampule broke while being introduced into the freeze-drying manifold and pierced the palm of the researcher. As a follow-up measure, the freeze drying procedure was reviewed and the pre-freezing temperature was reduced to −60°C. Measures were also taken to ensure use of better-quality ampules. Surgical and contact material associated with the lesion was placed in biohazard bags for autoclaving before disposal. In addition, laboratory and hospital staff was apprised of the risk associated with BPXV transmission. n nReporting of laboratory-acquired infections is crucial because infections could also spread to other personnel. Strict biosafety practices and laboratory guidelines are useful in minimizing laboratory-acquired infections. Guidelines, no matter how stringent, are not sufficient on their own. Laboratory-acquired infections occur because humans or machines are not infallible. Thus, laboratories should have emergency procedures in place to deal with such situations. n nTechnical Appendix: nPhylogenetic trees of A-type inclusion and C18L genes buffalopox virus and related viruses. n nClick here to view.(300K, pdf)

An Efficiently Cleaved HIV-1 Clade C Env Selectively Binds to Neutralizing Antibodies

An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design.

Sequence and phylogenetic analysis of host-range (E3L, K3L, and C7L) and structural protein (B5R) genes of buffalopox virus isolates from buffalo, cattle, and human in India

Buffalopox virus (BPXV), a close variant of vaccinia virus (VACV) has emerged as a zoonotic pathogen. The host tropism of poxviruses is governed by host-range genes. Among the host-range genes: E3L, K3L, and C7L are essential for virus replication by preventing interferon resistance, whereas B5R is essential for spread of the virus and evasion from the host’s immune response as in VACV. We report sequence analysis of host-range genes: E3L, K3L, C7L, and membrane protein gene (B5R) of BPXVs from buffalo, cattle, and human from recent outbreaks in India—their phylogenetic relationship with reference strain (BP4) and other Orthopoxviruses. BPXVs revealed a sequence homology with VACVs including zoonotic Brazilian VACV-like viruses. The aa sequences of E3L and K3L genes were 100xa0% similar in buffalo, cattle, and human isolates. However, four significant point mutations (I11K; N12K and S36F in C7L gene and D249G in B5R gene) were observed specific to buffalo isolate only. This signifies that different strains of BPXV were circulated during the outbreak. The mutations in C7L and B5R could play an important role in adaptation of BPXV in human and cattle which needs further functional studies. The strain of BPXV isolated from buffalo may not be adopted in human and cow. Various point mutations were observed in the host-range genes of reference strain (BPXV-BP4) which may be due to several passages of virus in cell culture. The phylogeny constructed based on concatenated gene sequences revealed that BPXVs are not as closely related to vaccine strain (Lister and Lister-derived strain—LC16m8), as hypothesized earlier, rather they are more closely related to reference strain (BPXV-BP4) and other vaccinia and vaccinia-like viruses such as Passatempo and Aracatuba viruses. The availability of information regarding host tropism determinants would allow us to understand molecular mechanism of species tropism of poxviruses which would be useful in unveiling new strategies to control zoonotic poxviral infections.

Membrane bound modified form of clade B Env, JRCSF is suitable for immunogen design as it is efficiently cleaved and displays all the broadly neutralizing epitopes including V2 and C2 domain-dependent conformational epitopes

BackgroundAntigenicity of HIV-1 envelope proteins (Envs) of both lab-adapted and primary isolates expressed on the cell surface rarely match with in vitro neutralization of viruses, pseudo-typed with corresponding Envs. Often, both neutralizing and non-neutralizing antibodies bind to Envs expressed on the cell membrane. This could be due to the lack of efficient cleavage of Env expressed on the cell surface. Naturally occurring, efficiently cleaved Envs with appropriate antigenic properties are relatively rare. Given viral diversity it is essential to increase the pool of candidate Envs suitable for immunogen design. Previously, it has been reported that JRFL Env is the only clade B Env, which is efficiently cleaved on the cell surface and retains desirable antigenic properties. JRCSF is a clade B Env isolated from the same patient as JRFL. JRCSF Env has not been explored aggressively for designing immunogen as the binding characteristics of JRCSF Env to broadly neutralizing antibodies on the cell surface and its cleavage status are unknown.ResultsAlthough JRCSF preferentially binds to most of the other gp120-directed neutralizing antibodies and cleavage dependent antibody, PGT151 efficiently, it binds poorly to CD4-binding-site-directed (CD4-bs-directed) neutralizing antibodies on cell surface. Membrane bound form of modified JRCSF Env containing the N197D mutation binds to CD4-bs-directed neutralizing antibodies better than JRFL, without debilitating its ability to bind quaternary epitope-directed neutralizing antibodies or exposing the CD4i antibody epitopes. In comparison to JRFL (E168K), JRCSF Env binds more efficiently to PG9/PGT145 class of V1/V2-directed conformational antibodies. Biochemical, cell surface staining and gp120 shedding experiments suggest that JRCSF is efficiently cleaved on the cell surface.ConclusionsBinding of JRCSF Env expressed on cell surface to the various HIV-1 Env-directed antibodies has not been reported earlier. Here, for the first time, we report that compared to JRFL, JRCSF displays epitopes for a larger number of broadly neutralizing antibodies and is also efficiently cleaved when expressed on the cell surface. Thus, considering the diversity of viral Envs and the discovery of conformation dependent glycan-directed antibodies in HIV-1 infected individuals, an innately cleaved JRCSF Env as present on the viral membrane and displaying those distinct epitopes may be an important candidate for immunogen design.

Identification and characterization of a naturally occurring, efficiently cleaved, membrane-bound, clade A HIV-1 Env, suitable for immunogen design, with properties comparable to membrane-bound BG505

Efficient cleavage of HIV-1 Env gp160 into its constituent subunits correlates with selective binding to neutralizing antibodies and are the closest mimetic of native, functional Envs. This was first demonstrated with the clade B Env, JRFL. The correlation between efficient cleavage and selective binding to neutralizing antibodies is the guiding principle for immunogen design for HIV vaccine. We have recently reported that Envs 4-2.J41 (clade C) and JRCSF (clade B) are also efficiently cleaved and show similar properties. However, an efficiently cleaved, membrane-bound clade A Env suitable for genetic vaccination has not been directly demonstrated. Here we report that BG505 and a new clade A Env, QB726.70M.ENV.C4 (or A5) are efficiently cleaved on cell membrane. A5 shows desirable antigenic properties comparable with BG505 on cell surface. A5SOSIP in supernatant displays majority of bNAb binding epitopes. Thus, both BG505 and A5 Envs can be used in DNA prime-protein boost vaccination studies.

Determinants in V2C2 region of HIV-1 clade C primary envelopes conferred altered neutralization susceptibilities to IgG1b12 and PG9 monoclonal antibodies in a context-dependent manner.

In the present study by examining pseudoviruses expressing patient chimeric envelopes (Envs) made between an IgG1b12 (b12)-sensitive (2-5.J3) and a b12-resistant (4.J22) HIV-1 clade C envelope, we identified determinants in the V2C2 region that governed susceptibility to b12 monoclonal antibody, but not to other CD4 binding site antibodies. Interestingly, when the V2C2 sequence of the 2-5.J3 Env was transferred to other b12-resistant primary clade C Envs, their susceptibility to b12 varied, indicating that this effect was context dependent. In addition, we identified determinants within the V2 region in the b12-resistant envelope that significantly modulated the neutralization of Env-pseudotyped viruses to PG9/PG16 MAbs. The enhanced neutralization susceptibilities of Envs to b12 and PG9 MAbs were correlated with increased exposure of their corresponding epitopes highlighting vulnerabilities in the V2C2 region that altered Env conformation necessary for the efficient accessibility of b12 and PG9 antibodies.

Cell surface ectodomain integrity of a subset of functional HIV-1 envelopes is dependent on a conserved hydrophilic domain containing region in their C-terminal tail

BackgroundHIV-1 Env gp160 is cleaved to form gp120 and gp41 and the functional HIV-1 Env is a trimer of non-covalently associated heterodimeric subunits, gp120 and gp41. The cleaved, native, trimeric form of Envs expose only broadly neutralizing antibody (bNAb) epitopes while occluding epitopes targeted by non-neutralizing antibodies (non-NAbs). We and others have previously observed that efficient cleavage of Envs into their constituent subunits co-relates with specific binding to bNAbs and poor binding to non-neutralizing antibodies (non-NAbs). Such Envs have been identified from clades A, B and C which make up a majority of globally circulating HIV-1 strains. Frequently, the C-terminal tail (CT) of Envs is deleted to enhance expression and stabilize soluble Env-based vaccine immunogens. Deletion of CT of efficiently cleaved Indian clade C Env 4-2.J41 results in recognition by both NAbs and non-NAbs. It is to be noted that uncleaved Envs bind to both NAbs and non-NAbs. So we investigated whether altered antigenicity upon CT deletion of efficiently cleaved Envs is due to inefficient cleavage or conformational change as the mechanism by which the CT regulates the ectodomain (ET) integrity is not well understood.ResultsWe studied the effect of CT deletion in four membrane bound efficiently cleaved Envs, A5 (clade A), 4-2.J41 (clade C), JRFL and JRCSF (clade B). Deletion of CT of the Envs, JRCSF and 4-2.J41, but not JRFL and A5 alter their ET antigenicity/conformation without affecting the cleavage efficiency. We carried out a series of deletion mutation in order to determine the region of the CT required for restoring native-like antigenicity/conformation of the ET of 4-2.J41 and JRCSF. Extending the CT up to aa753 in 4-2.J41 and aa759 in JRCSF, which includes a conserved hydrophilic domain (CHD), restores native-like conformation of these Envs on the plasma membrane. However, CT-deletion in 4-2.J41 and JRCSF at the pseudovirus level has either no or only modest effect on neutralization potency.ConclusionHere, we report that the CHD in the CT of Env plays an important role in regulating the ET integrity of a subset of efficiently cleaved, functional Envs on the cell surface.

Characterization of the membrane-bound form of the chimeric, B/C recombinant HIV-1 Env, LT5.J4b12C

Human immunodeficiency virus 1 (HIV-1) diversity is a significant challenge in developing a vaccine against the virus. B/C recombinants have been found in India and other places but are the predominant clade prevalent in China. HIV-1 envelopes (Envs) are the target of broadly neutralizing antibodies (bNAbs) which develop spontaneously in some HIV-1 infected patients. It has been previously reported with efficiently cleaved clade A, B and C Envs that preferential binding of Envs to bNAbs as opposed to non-NAbs, a desirable property for immunogens, is correlated with efficient cleavage of the Env precursor polypeptide into constituent subunits. These Envs are suitable for designing immunogens as soluble proteins, virus-like particles or for delivery by viral vectors/plasmid DNA. However, a B/C recombinant Env with similar properties has not been reported. Here we show that the chimeric, recombinant B/C clade Env LT5.J4b12C is efficiently cleaved on the plasma membrane and selectively binds to bNAbs.