Kennedy Epitope (KE)-dependent Retrograde Transport of Efficiently Cleaved HIV-1 Envelopes (Envs) and its Effect on Env Cell Surface Expression and Viral Particle Formation.

Efficiently cleaved HIV-1 Envs are the closest mimics of functional Envs as they specifically expose only bNAb (broadly neutralizing antibody) epitopes and not non-neutralizing ones, making them suitable for developing vaccine immunogens. We have previously identified several efficiently cleaved Envs from clades A, B, C and B/C. We also described that truncation of the CT (C-terminal tail) of a subset of these Envs, but not others, impairs their ectodomain conformation/antigenicity on the cell surface in a CT conserved hydrophilic domain (CHD) or Kennedy epitope (KE)-dependent manner. Here, we report that those Envs (4 - 2.J41 and JRCSF), whose native-like ectodomain conformation/antigenicity on the cell surface is disrupted upon CT truncation, but not other Envs like JRFL, whose CT truncation does not have an effect on ectodomain integrity on the cell surface, are also defective in retrograde transport from early to late endosomes. Restoration of the CHD/KE in the CT of these Envs restores wild-type levels of distribution between early and late endosomes. In the presence of retrograde transport inhibitor Retro 2, cell surface expression of 4 - 2.J41 and JRCSF Envs increases [as does in the presence of Rab7a DN and Rab7b DN (DN: dominant negative)] but particle formation decreases for 4 - 2.J41 and JRCSF Env pseudotyped viruses. Our results show for the first time a correlation between CT-dependent, CHD/KE regulated retrograde transport and cell surface expression/viral particle formation of these efficiently cleaved Envs. Based on our results we hypothesize that a subset of these efficiently cleaved Envs use a CT-dependent, CHD/KE-mediated mechanism for assembly and release from late endosomes.

A broadly neutralizing monoclonal antibody overcomes the mutational landscape of emerging SARS-CoV-2 variants of concern.

The emergence of new variants of SARS-CoV-2 necessitates unremitting efforts to discover novel therapeutic monoclonal antibodies (mAbs). Here, we report an extremely potent mAb named P4A2 that can neutralize all the circulating variants of concern (VOCs) with high efficiency, including the highly transmissible Omicron. The crystal structure of the P4A2 Fab:RBD complex revealed that the residues of the RBD that interact with P4A2 are a part of the ACE2-receptor-binding motif and are not mutated in any of the VOCs. The pan coronavirus pseudotyped neutralization assay confirmed that the P4A2 mAb is specific for SARS-CoV-2 and its VOCs. Passive administration of P4A2 to K18-hACE2 transgenic mice conferred protection, both prophylactically and therapeutically, against challenge with VOCs. Overall, our data shows that, the P4A2 mAb has immense therapeutic potential to neutralize the current circulating VOCs. Due to the overlap between the P4A2 epitope and ACE2 binding site on spike-RBD, P4A2 may also be highly effective against a number of future variants.

Biophysical and Biochemical Characterization of the Receptor Binding Domain of SARS-CoV-2 Variants.

The newly emerging SARS-CoV-2 variants are potential threat and posing new challenges for medical intervention due to high transmissibility and escaping neutralizing antibody (NAb) responses. Many of these variants have mutations in the receptor binding domain (RBD) of SARS-CoV-2 spike protein that interacts with the host cell receptor. Rapid mutation in the RBD through natural selection to improve affinity for host receptor and antibody pressure from vaccinated or infected individual will greatly impact the presently adopted strategies for developing interventions. Understanding the nature of mutations and how they impact the biophysical, biochemical and immunological properties of the RBD will help immensely to improve the intervention strategies. To understand the impact of mutation on the protease sensitivity, thermal stability, affinity for the receptor and immune response, we prepared several mutants of soluble RBD that belong to the variants of concern (VoCs) and interest (VoIs) and characterize them. Our results show that the mutations do not impact the overall structure of the RBD. However, the mutants showed increase in the thermal melting point, few mutants were more sensitive to protease degradation, most of them have enhanced affinity for ACE2 and some of them induced better immune response compared to the parental RBD.

Virus-Like Particles of SARS-CoV-2 as Virus Surrogates: Morphology, Immunogenicity, and Internalization in Neuronal Cells.

The engineering of virus-like particles (VLPs) is a viable strategy for the development of vaccines and for the identification of therapeutic targets without using live viruses. Here, we report the generation and characterization of quadruple-antigen SARS-CoV-2 VLPs. VLPs were generated by transient transfection of two expression cassettes in adherent HEK293T cells─one cassette containing Mpro for processing of three structural proteins (M, E, and N), and the second cassette expressing the Spike protein. Further characterization revealed that the VLPs retain close morphological and antigenic similarity with the native virus and also bind strongly to the SARS-CoV-2 receptor hACE-2 in an in vitro binding assay. Interestingly, the VLPs were found to internalize into U87-MG cells through cholesterol-rich domains in a dynamin-dependent process. Finally, our results showed that mice immunized with VLPs induce robust humoral and cellular immune responses mediated by enhanced levels of IL-4, IL-17, and IFNγ. Taken together, our results demonstrate that VLPs mimic the native virus and induce a strong immune response, indicating the possible use of these particles as an alternative vaccine candidate against SARS-CoV-2. VLPs can also be effective in mapping the initial stages of virus entry and screening inhibitors.

Characterization of a broadly cross reactive tetravalent human monoclonal antibody, recognizing conformational epitopes in receptor binding domain of SARS-CoV-2.

We used human semi-synthetic phage antibody gene libraries to select anti-SARS-CoV-2 RBD scFv antibody fragment and subsequent characterization of this novel tetravalent monoclonal antibody targeting conformational epitopes in the receptor binding domain of SARS-CoV-2. Binding studies suggest that II62 tetravalent antibody cross-reacts with RBD protein of SARS-CoV2 and its different variants of concerns. The epitope mapping data reveals that II62 tetravalent antibody targets an epitope that does not directly interferes with RBD: ACE2 interaction. Neutralization studies with live authentic SARS-CoV2 virus suggests that increase in valency of II62 mAb from monovalent to tetravalent doesn't perturbate virus interactions with the ACE2 expressing host cells in cytopathic effect-based (CPE) assay. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03272-6.

Protocol for High Throughput Screening of Antibody Phage Libraries.

Phage display is a proven and widely used technology for selecting specific antibodies against desired targets. However, an immense amount of effort is required to identify and screen the desired positive clones from large and diverse combinatorial libraries. On the other hand, the selection of positive binding clones from synthetic and semi-synthetic libraries has an inherent bias toward clones with randomly produced amber stop codons, making it more difficult to identify desirable binding antibodies. To overcome the screening of desired clones with amber codons, we present a step-by-step approach for effective phage library screening to isolate useful antibodies. The procedure calls for creating a simple new vector system for soluble production of phage ELISA positive binding clones with one or more amber stop codons in their single-chain antibody fragment (scFv) gene sequences, which is otherwise difficult in standard screening. Graphical abstract.

Generation of soluble, cleaved, well-ordered, native-like dimers of dengue virus 4 envelope protein ectodomain (sE) suitable for vaccine immunogen design.

Dengue virus is transmitted by Aedes mosquitoes and dengue is endemic in many regions of the world. Severe dengue results in complications that may lead to death. Although some vaccine candidates are in clinical trials and one vaccine Dengvaxia, with restricted efficacy, is available, there are currently no specific therapies to completely prevent or treat dengue. The dengue virus structural protein E (envelope) exists as a head-to-tail dimer on mature virus, is targeted by broadly neutralizing antibodies and is suitable for developing vaccine immunogens. Here, we have used a redesigned dengue prME expression construct and immunoaffinity chromatography with conformational/quaternary antibody A11 to purify soluble DENV4 sE(A259C) (E ectodomain) dimers from mammalian expression system to ~99 % purity. These dimers retain glycosylation reported for native DENV E, display the three major broadly neutralizing antibody epitopes, and form well-ordered structure. This strategy can be used for developing subunit vaccine candidates against dengue and other flaviviruses.

Inactivated whole-virion vaccine BBV152/Covaxin elicits robust cellular immune memory to SARS-CoV-2 and variants of concern.

BBV152 is a whole-virion inactivated vaccine based on the Asp614Gly variant. BBV152 is the first alum-imidazoquinolin-adjuvanted vaccine authorized for use in large populations. Here we characterized the magnitude, quality and persistence of cellular and humoral memory responses up to 6 months post vaccination. We report that the magnitude of vaccine-induced spike and nucleoprotein antibodies was comparable with that produced after infection. Receptor binding domain-specific antibodies declined against variants in the order of Alpha (B.1.1.7; 3-fold), Delta (B.1.617.2; 7-fold) and Beta (B.1.351; 10-fold). However, pseudovirus neutralizing antibodies declined up to 2-fold against the Delta followed by the Beta variant (1.7-fold). Vaccine-induced memory B cells were also affected by the Delta and Beta variants. The SARS-CoV-2-specific multicytokine-expressing CD4+ T cells were found in ~85% of vaccinated individuals. Only a ~1.3-fold reduction in efficacy was observed in CD4+ T cells against the Beta variant. We found that antigen-specific CD4+ T cells were present in the central memory compartment and persisted for at least up to 6 months post vaccination. Vaccine-induced CD8+ T cells were detected in ~50% of individuals. Importantly, the vaccine was capable of inducing follicular T helper cells that exhibited B-cell help potential. These findings show that inactivated vaccine BBV152 induces robust immune memory to SARS-CoV-2 and variants of concern that persists for at least 6 months after vaccination.

Designing and characterization of a SARS-CoV-2 immunogen with receptor binding motif grafted on a protein scaffold: An epitope-focused vaccine approach.

The COVID-19 pandemic caused by SARS-CoV-2 has a significant burden on the economy and healthcare around the world. Vaccines are the most effective tools to fight infectious diseases by containing the spread of the disease. The current vaccines against SARS-CoV-2 are mostly based on the spike protein of SARS-CoV-2, which is large and has many immune-dominant non-neutralizing epitopes that may effectively skew the antibody response towards non-neutralizing antibodies. Here, we have explored the possibility of immune-focusing the receptor binding motif (RBM) of the spike protein of SARS-CoV-2 that induces mostly neutralizing antibodies in natural infection or in vacinees. The result shows that the scaffolded RBM can bind to Angiotensin Converting Enzyme 2 (ACE2) although with low affinity and induces a strong antibody response in mice. The immunized sera can bind both, the receptor binding domain (RBD) and the spike protein, which holds the RBM in its natural context. Sera from the immunized mice showed robust interferon γ response but poor neutralization of SARS-CoV-2 suggesting presence of a predominant T cell epitope on scaffolded RBM. Together, we provide a strategy for inducing strong antigenic T cell response which could be exploited further for future vaccine designing and development against SARS-CoV-2 infection.

The SARS CoV-2 spike directed non-neutralizing polyclonal antibodies cross-react with Human immunodeficiency virus (HIV-1) gp41.

Cross-reactivity among the two diverse viruses is believed to originate from the concept of antibodies recognizing similar epitopes on the two viral surfaces. Cross-reactive antibody responses have been seen in previous variants of SARS and SARS-CoV-2, but little is known about the cross reactivity with other similar RNA viruses like HIV-1. In the present study, we examined the reactivity the SARS-CoV-2 directed antibodies, via spike, immunized mice sera and demonstrated whether they conferred any cross-reactive neutralization against HIV-1. Our findings show that SARS-CoV-2 spike immunized mice antibodies cross-react with the HIV-1 Env protein. Cross-neutralization among the two viruses is uncommon, suggesting the presence of a non-neutralizing antibody response to conserved epitopes amongst the two viruses. Our results indicate, that SARS-CoV-2 spike antibody cross reactivity is targeted towards the gp41 region of the HIV-1 Env (gp160) protein. Overall, our investigation not only answers a crucial question about the understanding of cross-reactive epitopes of antibodies generated in different viral infections, but also provides critical evidence for developing vaccine immunogens and novel treatment strategies with enhanced efficacy capable of recognising diverse pathogens with similar antigenic features.

Chikungunya and arthritis: An overview.

Chikungunya is caused by CHIKV (chikungunya virus), an emerging and re-emerging arthropod-vectored viral infection that causes a febrile disease with primarily long term sequelae of arthralgia and myalgia and is fatal in a small fraction of infected patients. Sporadic outbreaks have been reported from different parts of the world chiefly Africa, Asia, the Indian and Pacific ocean regions, Europe and lately even in the Americas. Currently, treatment is primarily symptomatic as no vaccine, antibody-mediated immunotherapy or antivirals are available. Chikungunya belongs to a family of arthritogenic alphaviruses which have many pathophysiological similarities. Chikungunya arthritis has similarities and differences with rheumatoid arthritis. Although research into arthritis caused by these alphaviruses have been ongoing for decades and significant progress has been made, the mechanisms underlying viral infection and arthritis are not well understood. In this review, we give a background to chikungunya and the causative virus, outline the history of alphavirus arthritis research and then give an overview of findings on arthritis caused by CHIKV. We also discuss treatment options and the research done so far on various therapeutic intervention strategies.

Inhalation monoclonal antibody therapy: a new way to treat and manage respiratory infections.

The route of administration of a therapeutic agent has a substantial impact on its success. Therapeutic antibodies are usually administered systemically, either directly by intravenous route, or indirectly by intramuscular or subcutaneous injection. However, treatment of diseases contained within a specific tissue necessitates a better alternate route of administration for targeting localised infections. Inhalation is a promising non-invasive strategy for antibody delivery to treat respiratory maladies because it provides higher concentrations of antibody in the respiratory airways overcoming the constraints of entry through systemic circulation and uncertainity in the amount reaching the target tissue. The nasal drug delivery route is one of the extensively researched modes of administration, and nasal sprays for molecular drugs are deemed successful and are presently commercially marketed. This review highlights the current state and future prospects of inhaled therapies, with an emphasis on the use of monoclonal antibodies for the treatment of respiratory infections, as well as an overview of their importance, practical challenges, and clinical trial outcomes.Key points• Immunologic strategies for preventing mucosal transmission of respiratory pathogens.• Mucosal-mediated immunoprophylaxis could play a major role in COVID-19 prevention.• Applications of monoclonal antibodies in passive immunisation.

Non-neutralizing SARS CoV-2 directed polyclonal antibodies demonstrate cross-reactivity with the HA glycans of influenza virus.

The spike protein of the SARS-CoV-2 virus is the foremost target for the designing of vaccines and therapeutic antibodies and also acts as a crucial antigen in the assessment of COVID-19 immune responses. The enveloped viruses; such as SARS-CoV-2, Human Immunodeficiency Virus-1 (HIV-1) and influenza, often hijack host-cell glycosylation pathways and influence pathobiology and immune selection. These glycan motifs can lead to either immune evasion or viral neutralization by the production of cross-reactive antibodies that can lead to antibody-dependent enhancement (ADE) of infection. Potential cross-protection from influenza vaccine has also been reported in COVID-19 infected individuals in several epidemiological studies recently; however, the scientific basis for these observations remains elusive. Herein, we show that the anti-SARS-CoV2 antibodies cross-reacts with the Hemagglutinin (HA) protein. This phenomenon is common to both the sera from convalescent SARS-CoV-2 donors and spike immunized mice, although these antibodies were unable to cross-neutralize, suggesting the presence of a non-neutralizing antibody response. Epitope mapping suggests that the cross-reactive antibodies are targeted towards glycan epitopes of the SARS-CoV-2 spike and HA. Overall, our findings address the cross-reactive responses, although non-neutralizing, elicited against RNA viruses and warrant further studies to investigate whether such non-neutralizing antibody responses can contribute to effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or ADE.

A rapid novel strategy for screening of antibody phage libraries for production, purification, and functional characterization of amber stop codons containing single-chain antibody fragments.

Phage display antibody (PDA) libraries, allows the rapid isolation and characterization of high specificity monoclonal antibodies for therapeutic and diagnostic applications. However, selection of positive binding clones from synthetic and semi-synthetic libraries has an inherent bias towards clones containing randomly generated amber stop codons, complicating the identification of high affinity binding antibodies. We screened Tomlinson I and J library against receptor binding domain (RBD) of SARS CoV2, eight clones which showed positive binding in phage ELISA, contained one or more amber stop codons in their single-chain antibody fragment (scFv) gene sequences. The presence of amber stop codons within the antibody sequence causes the premature termination of soluble form of scFv expression in nonsuppressor Escherichia coli strain. In the present study, we have used a novel strategy that allows soluble expression of scFvs having amber stop codon in their gene sequences (without phage PIII protein fusion), in the suppressor strain. This strategy of introduction of Ochre (TAA) codon at the junction of scFv and PIII gene, speeds up the initial screening process which is critical for selecting the right scFvs for further studies. Present strategy leads to the identification of a scFv, B8 that binds specifically with nanomolar affinity toward SARS CoV 2 RBD, which otherwise lost in terms of traditional methodology.

Efficiently cleaved HIV-1 envelopes: can they be important for vaccine immunogen development?

The enormous diversity of HIV-1 is a significant impediment in selecting envelopes (Envs) that can be suitable for designing vaccine immunogens. While tremendous progress has been made in developing soluble, trimeric, native-like Env proteins, those that have elicited neutralizing antibodies (Abs) in animal models are relatively few. A strategy of selecting naturally occurring Envs suitable for immunogen design by studying the correlation between efficient cleavage on the cell surface and their selective binding to broadly neutralizing Abs (bNAbs) and not to non-neutralizing Abs (non-NAbs), properties essential in immunogens, may be useful. Here we discuss some of the challenges of developing an efficacious HIV-1 vaccine and the work done in generating soluble immunogens. We also discuss the study of naturally occurring, membrane-bound, efficiently cleaved (naturally more sensitive to furin) Envs and how they may positively add to the repertoire of HIV-1 Envs that can be used for vaccine immunogen design. However, even with such Envs, the challenges of developing well-folded, native-like trimers as soluble proteins or using other immunogen strategies such as virus-like particles with desirable antigenic properties remain, and are formidable. In spite of the progress that has been made in the HIV-1 vaccine field, an immunogen that elicits neutralizing Abs with significant breadth and potency in vaccines has still not been developed. Efficiently cleaved Envs may increase the number of available Envs suitable for immunogen design and should be studied further.

Identification of an anti-SARS-CoV-2 receptor-binding domain-directed human monoclonal antibody from a naïve semisynthetic library.

There is a desperate need for safe and effective vaccines, therapies, and diagnostics for SARS- coronavirus 2 (CoV-2), the development of which will be aided by the discovery of potent and selective antibodies against relevant viral epitopes. Human phage display technology has revolutionized the process of identifying and optimizing antibodies, providing facile entry points for further applications. Herein, we use this technology to search for antibodies targeting the receptor-binding domain (RBD) of CoV-2. Specifically, we screened a naïve human semisynthetic phage library against RBD, leading to the identification of a high-affinity single-chain fragment variable region (scFv). The scFv was further engineered into two other antibody formats (scFv-Fc and IgG1). All three antibody formats showed high binding specificity to CoV-2 RBD and the spike antigens in different assay systems. Flow cytometry analysis demonstrated specific binding of the IgG1 format to cells expressing membrane-bound CoV-2 spike protein. Docking studies revealed that the scFv recognizes an epitope that partially overlaps with angiotensin-converting enzyme 2 (ACE2)-interacting sites on the CoV-2 RBD. Given its high specificity and affinity, we anticipate that these anti-CoV-2 antibodies will be useful as valuable reagents for accessing the antigenicity of vaccine candidates, as well as developing antibody-based therapeutics and diagnostics for CoV-2.

Hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives.

The advancements in technology and manufacturing processes have allowed the development of new derivatives, biosimilar or advanced improved versions for approved antibodies each year for treatment regimen. There are more than 700 antibody-based molecules that are in different stages of phase I/II/ III clinical trials targeting new unique targets. To date, approximately more than 80 monoclonal antibodies (mAbs) have been approved. A total of 7 novel antibody therapeutics had been granted the first approval either in the United States or European Union in the year 2019, representing approximately 20% of the total number of approved drugs. Most of these licenced mAbs or their derivatives are either of hybridoma origin or their improvised engineered versions. Even with the recent development of high throughput mAb generation technologies, hybridoma is the most favoured method due to its indigenous nature to preserve natural cognate antibody pairing information and preserves innate functions of immune cells. The recent advent of antibody engineering technology has superseded the species level barriers and has shown success in isolation of hybridoma across phylogenetically distinct species. This has led to the isolation of monoclonal antibodies against human targets that are conserved and non-immunogenic in the rodent. In this review, we have discussed in detail about hybridoma technology, its expansion towards different animal species, the importance of antibodies isolated from different animal sources that are useful in biological applications, advantages, and limitations. This review also summarizes the challenges and recent progress associated with hybridoma development, and how it has been overcome in these years to provide new insights for the isolation of mAbs.

Antibody-based therapeutic interventions: possible strategy to counter chikungunya viral infection.

Chikungunya virus (CHIKV), a mosquito-transmitted disease that belongs to the genus Alphaviruses, has been emerged as an epidemic threat over the last two decades, and the recent co-emergence of this virus along with other circulating arboviruses and comorbidities has influenced atypical mortality rate up to 10%. Genetic variation in the virus has resulted in its adaptability towards the new vector Aedes albopictus other than Aedes aegypti, which has widen the horizon of distribution towards non-tropical and non-endemic areas. As of now, no licensed vaccines or therapies are available against CHIKV; the treatment regimens for CHIKV are mostly symptomatic, based on the clinical manifestations. Development of small molecule drugs and neutralizing antibodies are potential alternatives of worth investigating until an efficient or safe vaccine is approved. Neutralizing antibodies play an important role in antiviral immunity, and their presence is a hallmark of viral infection. In this review, we describe prospects for effective vaccines and highlight importance of neutralizing antibody-based therapeutic and prophylactic applications to combat CHIKV infections. We further discuss about the progress made towards CHIKV therapeutic interventions as well as challenges and limitation associated with the vaccine development. Furthermore this review describes the lesson learned from chikungunya natural infection, which could help in better understanding for future development of antibody-based therapeutic measures.

Phage display antibody libraries: A robust approach for generation of recombinant human monoclonal antibodies.

Monoclonal antibodies (mAbs) and their derivatives have achieved remarkable success as medicine, targeting both diagnostic and therapeutic applications associated with communicable and non-communicable diseases. In the last 3 to 4 decades, tremendous success has been manifested in the field of cancer therapy, autoimmune diseases, cardiovascular and infectious diseases. MAbs are the fastest growing class of biopharmaceuticals, with more than 25 derivatives are in clinical use and 7 of these have been isolated through phage display technology. Phage display technology has gained impetus in the field of medical and health sciences, as a large repertoire of diverse recombinant antibodies, targeting various antigens have been generated in a short span of time. A prominent number of phage display derived antibodies are already approved for therapy and significant numbers are currently in clinical trials. In this review we have discussed the various strategies employed for generation of monoclonal antibodies; their advantages, limitations and potential therapeutic applications. We also discuss the potential of phage display antibody libraries in isolation of monoclonal antibodies.