Antivirals Targeting the Neuraminidase.

The neuraminidase (NA) of influenza A and B viruses plays a distinct role in viral replication and has a highly conserved catalytic site. Numerous sialic (neuraminic) acid analogs that competitively bind to the NA active site and potently inhibit enzyme activity have been synthesized and tested. Four NA inhibitors are now licensed in various parts of the world (zanamivir, oseltamivir, peramivir, and laninamivir) to treat influenza A and B infections. NA changes, naturally occurring or acquired under selective pressure, have been shown to reduce drug binding, thereby affecting the effectiveness of NA inhibitors. Drug resistance and other drawbacks have prompted the search for the next-generation NA-targeting therapeutics. One of the promising approaches is the identification of monoclonal antibodies (mAbs) targeting the conserved NA epitopes. Anti-NA mAbs demonstrate Fab-based antiviral activity supplemented with Fc-mediated immune effector functions. Antiviral Fc-conjugates offer another cutting-edge strategy that is based on a multimodal mechanism of action. These novel antiviral agents are composed of a small-molecule NA inhibitor and an Fc-region that simultaneously engages the immune system. The significant advancements made in recent years further support the value of NA as an attractive target for the antiviral development.

Cluster of Oseltamivir-Resistant and Hemagglutinin Antigenically Drifted Influenza A(H1N1)pdm09 Viruses, Texas, USA, January 2020.

Four cases of oseltamivir-resistant influenza A(H1N1)pdm09 virus infection were detected among inhabitants of a border detention center in Texas, USA. Hemagglutinin of these viruses belongs to 6B.1A5A-156K subclade, which may enable viral escape from preexisting immunity. Our finding highlights the necessity to monitor both drug resistance and antigenic drift of circulating viruses.

Sequential immunizations with a panel of HIV-1 Env virus-like particles coach immune system to make broadly neutralizing antibodies.

Broadly neutralizing antibodies (bnAbs) are correlated with passive HIV/SHIV protection and are desirable components of a HIV protective immunity. In the current study, we have designed a sequential-immunization strategy with a panel of envelope glycoprotein (Env)-enriched virus-like particles (VLPs) from various HIV-1 clades (A-E) to elicit bnAbs with high breadth and potency of neutralization in rabbits. We have compared this regimen with repetitive immunizations of individual Env (subtype B) VLPs or a mixture of various Env VLPs. Our results demonstrate that the sequential immunization group of animals induced significantly higher IgG endpoint titers against respective HIV Env (autologous) antigen than other control groups. Animals vaccinated sequentially showed an increase in the antibody endpoint titers and IgG antibody secreting cells (ASCs) against Con-S Env protein. Sequential immunizations with various Env VLPs promoted antibody avidity indices and enhanced bnAb responses against a panel of HIV pseudotyped virions including some of the tier 3 pseudostrains. Sequential immunizations with various VLPs displaying "native-like" HIV-1 Envs elicited bnAb responses with increased breadth and potency of neutralization.

Sequential Immunizations with heterosubtypic virus-like particles elicit cross protection against divergent influenza A viruses in mice.

Seasonal influenza vaccines have proven to be effective against well-matched viruses in healthy adults. However, rapid accumulation of mutations in the main antigenic surface proteins of influenza can compromise the efficiency of flu vaccines. Occasionally, influenza pandemics arise and present a different type of challenge to current seasonal vaccines. Novel vaccination strategies that can educate the host immune system to generate immune responses focusing on conserved epitopes on theses antigenic surface proteins are crucial for controlling and limiting influenza epidemics and pandemics. In this study, we have sequentially vaccinated mice with heterosubtypic influenza HA virus-like particles (VLPs) harboring H1, H8, and H13 from the HA phylogenetic group 1, or H3, H4, and H10 from the HA phylogenetic group 2, or in various combinations. The immunized animals were fully protected when challenged with lethal doses of heterosubtypic viruses from either phylogenetic group. Our vaccination approach demonstrates a promising strategy for the development of a 'universal influenza vaccine'.

Double-layered protein nanoparticles induce broad protection against divergent influenza A viruses.

Current influenza vaccines provide limited protection against circulating influenza A viruses. A universal influenza vaccine will eliminate the intrinsic limitations of the seasonal flu vaccines. Here we report methodology to generate double-layered protein nanoparticles as a universal influenza vaccine. Layered nanoparticles are fabricated by desolvating tetrameric M2e into protein nanoparticle cores and coating these cores by crosslinking headless HAs. Representative headless HAs of two HA phylogenetic groups are constructed and purified. Vaccinations with the resulting protein nanoparticles in mice induces robust long-lasting immunity, fully protecting the mice against challenges by divergent influenza A viruses of the same group or both groups. The results demonstrate the importance of incorporating both structure-stabilized HA stalk domains and M2e into a universal influenza vaccine to improve its protective potency and breadth. These potent disassemblable protein nanoparticles indicate a wide application in protein drug delivery and controlled release.

Applications of chemokines as adjuvants for vaccine immunotherapy.

Vaccinations are expected to aid in building immunity against pathogens. This objective often requires the addition of an adjuvant with certain vaccine formulations containing weakly immunogenic antigens. Adjuvants can improve antigen processing, presentation, and recognition, thereby improving the immunogenicity of a vaccine by simulating and eliciting an immune response. Chemokines are a group of small chemoattractant proteins that are essential regulators of the immune system. They are involved in almost every aspect of tumorigenesis, antitumor immunity, and antimicrobial activity and also play a critical role in regulating innate and adaptive immune responses. More recently, chemokines have been used as vaccine adjuvants due to their ability to modulate lymphocyte development, priming and effector functions, and enhance protective immunity. Chemokines that are produced naturally by the body's own immune system could serve as potentially safer and more reliable adjuvant options versus synthetic adjuvants. This review will primarily focus on chemokines and their immunomodulatory activities against various infectious diseases and cancers.

CCL28 chemokine: An anchoring point bridging innate and adaptive immunity.

Chemokines are an extensive family of small proteins which, in conjunction with their receptors, guide the chemotactic activity of various immune cells throughout the body. CCL28, ß- or CC chemokine, is involved in the host immunity at various epithelial and mucosal linings. The unique roles of CCL28 in several facets of immune responses have attracted considerable attention and may represent a promising approach to combat various infections. CCL28 displays a broad spectrum of antimicrobial activity against gram-negative and gram-positive bacteria, as well as fungi. Here, we will summarize various research findings regarding the antimicrobial activity of CCL28 and the relevant mechanisms behind it. We will explore how the structure of CCL28 is involved with this activity and how this function may have evolved. CCL28 displays strong homing capabilities for B and T cells at several mucosal and epithelial sites, and orchestrates the trafficking and functioning of lymphocytes. The chemotactic and immunomodulatory features of CCL28 through the interactions with its chemokine receptors, CCR10 and CCR3, will also be discussed in detail. Thus, in this review, we emphasize the dual properties of CCL28 and suggest its role as an anchoring point bridging the innate and adaptive immunity.

Protein nanoparticle vaccine based on flagellin carrier fused to influenza conserved epitopes confers full protection against influenza A virus challenge.

Currently marketed influenza vaccines only confer protection against matching influenza virus strains. The influenza A composition of these vaccines needs to be annually updated. Vaccines that target conserved epitopes of influenza viruses would in principle offer broad cross-protection against influenza A viruses. In our study, we investigated the specific immune responses and protective efficacy of protein nanoparticles based on fusion proteins of flagellin carrier linked to conserved influenza epitopes. We designed fusion proteins by replacing the hyperimmunogenic region of flagellin (FliC) with four tandem copies of the ectodomain of matrix protein 2 (f4M2e), H1 HA2 domain (fHApr8) or H3 HA2 domain (fHAaichi). Protein nanoparticles fabricated from these fusion proteins by using DTSSP crosslinking retained Toll-like receptor 5 agonist activity of FliC. Intranasal immunization with f4M2e, f4M2e/fHApr8 or f4M2e/fHAaichi nanoparticles induced vaccine antigen-specific humoral immune responses. It was also found that the incorporation of the H1 HA2 domain into f4M2e/fHApr8 nanoparticles boosted M2e specific antibody responses. Immunized mice were fully protected against lethal doses of virus challenge.

A boosting skin vaccination with dissolving microneedle patch encapsulating M2e vaccine broadens the protective efficacy of conventional influenza vaccines.

The biodegradable microneedle patch (MNP) is a novel technology for vaccine delivery that could improve the immunogenicity of vaccines. To broaden the protective efficiency of conventional influenza vaccines, a new 4M2e-tFliC fusion protein construct containing M2e sequences from different subtypes was generated. Purified fusion protein was encapsulate into MNPs with a biocompatible polymer for use as a boosting vaccine. The results demonstrated that mice receiving a conventional inactivated vaccine followed by a skin-applied dissolving 4M2e-tFliC MNP boost could better maintain the humoral antibody response than that by the conventional vaccine-prime alone. Compared with an intramuscular injection boost, mice receiving the MNP boost showed significantly enhanced cellular immune responses, hemagglutination-inhibition (HAI) titers, and neutralization titers. Increased frequency of antigen-specific plasma cells and long-lived bone marrow plasma cells was detected in the MNP boosted group as well, indicating that skin vaccination with 4M2e-tFliC facilitated a long-term antibody-mediated immunity. The 4M2e-tFliC MNP-boosted group also possessed enhanced protection against high lethal dose challenges against homologous A/PR/8/34 and A/Aichi/2/68 viruses and protection for a majority of immunized mice against a heterologous A/California/07/2009 H1N1 virus. High levels of M2e specific immune responses were observed in the 4M2e-tFliC MNP-boosted group as well. These results demonstrate that a skin-applied 4M2e-tFliC MNP boosting immunization to seasonal vaccine recipients may be a rapid approach for increasing the protective efficacy of seasonal vaccines in response to a significant drift seen in circulating viruses. The results also provide a new perspective for future exploration of universal influenza vaccines.

Chimeric virus-like particles containing influenza HA antigen and GPI-CCL28 induce long-lasting mucosal immunity against H3N2 viruses.

Influenza virus is a significant cause of morbidity and mortality, with worldwide seasonal epidemics. The duration and quality of humoral immunity and generation of immunological memory to vaccines is critical for protective immunity. In the current study, we examined the long-lasting protective efficacy of chimeric VLPs (cVLPs) containing influenza HA and GPI-anchored CCL28 as antigen and mucosal adjuvant, respectively, when immunized intranasally in mice. We report that the cVLPs induced significantly higher and sustainable levels of virus-specific antibody responses, especially IgA levels and hemagglutination inhibition (HAI) titers, more than 8-month post-vaccination compared to influenza VLPs without CCL28 or influenza VLPs physically mixed with sCCL28 (soluble) in mice. After challenging the vaccinated animals at month 8 with H3N2 viruses, the cVLP group also demonstrated strong recall responses. On day 4 post-challenge, we measured increased antibody levels, ASCs and HAI titers with reduced viral load and inflammatory responses in the cVLP group. The animals vaccinated with the cVLP showed 20% cross-protection against drifted (Philippines) and 60% protection against homologous (Aichi) H3N2 viruses. Thus, the results suggest that the GPI-anchored CCL28 induces significantly higher mucosal antibody responses, involved in providing long-term cross-protection against H3N2 influenza virus when compared to other vaccination groups.

Coated protein nanoclusters from influenza H7N9 HA are highly immunogenic and induce robust protective immunity.

Recurring influenza viruses pose an annual threat to public health. A time-saving, cost-effective and egg-independent influenza vaccine approach is important particularly when responding to an emerging pandemic. We fabricated coated, two-layer protein nanoclusters from recombinant trimeric hemagglutinin from an avian-origin H7N9 influenza A virus as an approach for vaccine development in response to an emerging pandemic. Assessment of the virus-specific immune responses and protective efficacy in mice immunized with the nanoclusters demonstrated that the vaccine candidates were highly immunogenic, able to induce protective immunity and long-lasting humoral antibody responses to this virus without the use of adjuvants. Because the advantages of the highly immunogenic coated nanoclusters also include rapid productions in an egg-independent system, this approach has great potential for influenza vaccine production not only in response to an emerging pandemic, but also as a replacement for conventional seasonal influenza vaccines.

Coexistence of Th1/Th2 and Th17/Treg imbalances in patients with post traumatic sepsis.

INTRODUCTION: Multiple organ dysfunction syndrome (MODS) developed due to the insult of trauma is a leading cause of death. The high mortality rate in these patients with and without sepsis has been reported up to 50%, throughout the world and thus required an urgent insight to overcome this problem. OBJECTIVE: The aim of this study is to examine the differential changes in subsets of T cells, imbalance in cytokine profile, immune-paralysis (T cell anergy) in Trauma hemorrhagic shock (THS) and post traumatic sepsis patients. METHODOLOGY: 114, THS patients and 50 healthy controls were recruited in the present study. We have measured the T cell proliferation assay using dominant antigens of both gram positive (LTA, 100ng/ml) and gram negative (LPS-100ng/ml) bacteria and PHA (4µg/ml) using radioactive thymidine (1H3) assay. Simultaneously, we have measured the culture supernatant level of cytokines using Cytokine bead assay (CBA). The other parts of this study include the analysis of different subsets of T cells. RESULTS AND CONCLUSION: We observed significantly (P<0.05) reduced T cell proliferation in THS patients as compared to control. Our study also showed patients died due to sepsis/septic shock, had significantly (p<0.05) lower T cell response and had significantly elevated levels of IL-4, IL-10andTGF-ß, but low level of IL-2andIFN-γ in culture supernatant. THS patients who developed sepsis complication had significantly higher T regulatory cells and lower Th17 cells in comparison to non-sepsis. In conclusion, our study showed an imbalance in cell mediated immune response and disturbance in Th1/Th2/Th17 and T reg population of T helper cells and also the shifts towards Th2 and T17 in THS patients who had developed sepsis and showed poor outcomes.

Co-delivery of GPI-anchored CCL28 and influenza HA in chimeric virus-like particles induces cross-protective immunity against H3N2 viruses.

Influenza infection typically initiates at respiratory mucosal surfaces. Induction of immune responses at the sites where pathogens initiate replication is crucial for the prevention of infection. We studied the adjuvanticity of GPI-anchored CCL28 co-incorporated with influenza HA-antigens in chimeric virus-like particles (cVLPs), in boosting strong protective immune responses through an intranasal (i.n.) route in mice. We compared the immune responses to that from influenza VLPs without CCL28, or physically mixed with soluble CCL28 at systemic and various mucosal compartments. The cVLPs containing GPI-CCL28 showed in-vitro chemotactic activity towards spleen and lung cells expressing CCR3/CCR10 chemokine receptors. The cVLPs induced antigen specific endpoint titers and avidity indices of IgG in sera and IgA in tracheal, lung, and intestinal secretions, significantly higher (4-6 fold) than other formulations. Significantly higher (3-5 fold) hemagglutination inhibition titers and high serum neutralization against H3N2 viruses were also detected with CCL28-containing VLPs compared to other groups. The CCL28-containing VLPs showed complete and 80% protection, when vaccinated animals were challenged with A/Aichi/2/1968/H3N2 (homologous) and A/Philippines/2/1982/H3N2 (heterologous) viruses, respectively. Thus, GPI-anchored CCL28 in influenza VLPs act as a strong immunostimulator at both systemic and mucosal sites, boosting significant cross-protection in animals against heterologous viruses across a large distance.

Evaluation of multiple antigenic peptides based on the Chikungunya E2 protein for improved serological diagnosis of infection.

In recent years, Chikungunya virus (CHIKV) reemerged and numerous outbreaks were reported all over the world. After screening CHIKV-positive sera, we had already reported many dominant epitopes within the envelope E2 protein of CHIKV. In the present study, we aimed at developing a highly sensitive immunodiagnostic assay for CHIKV based on a multiple antigenic peptide (MAP) approach using selective epitopes of the E2 protein. MAPs in four different E2 peptide combinations were screened with CHIKV-positive sera. The MAPs reacted with all CHIKV-positive sera and no reactivity was seen with healthy or dengue-positive sera. Our results indicate that MAP 1 seems to be an alternate antigen to full-length protein E2 for immunodiagnosis of CHIKV infections with high sensitivity and specificity.

Current understanding of HIV-1 and T-cell adaptive immunity: progress to date.

The cellular immune response to human immunodeficiency virus (HIV) has different components originating from both the adaptive and innate immune systems. HIV cleverly utilizes the host machinery to survive by its intricate nature of interaction with the host immune system. HIV evades the host immune system at innate ad adaptive, allows the pathogen to replicate and transmit from one host to another. Researchers have shown that HIV has multipronged effects especially on the adaptive immunity, with CD4(+) cells being the worst effect T-cell populations. Various analyses have revealed that, the exposure to HIV results in clonal expansion and excessive activation of the immune system. Also, an abnormal process of differentiation has been observed suggestive of an alteration and blocks in the maturation of various T-cell subsets. Additionally, HIV has shown to accelerate immunosenescence and exhaustion of the overtly activated T-cells. Apart from causing phenotypic changes, HIV has adverse effects on the functional aspect of the immune system, with evidences implicating it in the loss of the capacity of T-cells to secrete various antiviral cytokines and chemokines. However, there continues to be many aspects of the immune- pathogenesis of HIV that are still unknown and thus required further research in order to convert the malaise of HIV into a manageable epidemic.

Nasal delivery of PLG microparticle encapsulated defensin peptides adjuvanted gp41 antigen confers strong and long-lasting immunoprotective response against HIV-1.

Defensins display immunostimulatory activities including a chemotactic effect for T lymphocytes/immature dendritic cells and secretion of pro-inflammatory cytokines suggest their role in bridging innate and adaptive immunity. We hypothesized whether defensins with separately emulsified HIV-1 immunogen would elicit peptide-specific systemic and mucosal antibody response in mice. The HIV-1 peptide alone in microsphere showed low peptide-specific antibody response in sera and different washes, while the presence of defensins markedly increased the antibody peak titre both in sera (102,400-409,600) (p < 0.05) and in washes (800-25,600) (p < 0.001). Defensins with HIV-1 peptide were showing 43.0-83.2% and 38.7-72.3% in vitro neutralization against laboratory isolates in serum and lavage samples, respectively, higher than HIV-1 peptide alone. Our findings may have implications in the development of new mucosal adjuvant for AIDS vaccination.

Comparative mucosal immunogenicity of HIV gp41 membrane-proximal external region (MPER) containing single and multiple repeats of ELDKWA sequence with defensin peptides.

The MPER of gp41 of HIV-1 has received great attention and is widely recognized as a promising target for the development of AIDS vaccine. We investigated the ability of trirepeat of ELDKWA sequence of gp41 antigen with defensins in liposome using multiple-shot immunization strategy in the mice model. The designed was used to enhance the immunogenicity and exposure of MPER in its native conformation for the induction of MPER-specific HIV-1 neutralizing antibodies. To characterize, we estimated the antibody levels (IgG/IgA) in serum as well as in lung, intestinal, vaginal and rectal washes till day 120 in outbred and inbred (H-2(b) and H-2(d)) mice using liposome as delivery vehicle. The representative sera and washes were also tested for in vitro neutralization with CCR5-tropic Indian HIV-1 primary isolates. We observed that the modified HIV antigen containing trirepeat of ELDKWA with defensins was showing significantly (p<0.001) higher IgG/IgA antibody titre (102,400-204,800) in sera as well as in different mucosal washes (1600-6400) than standard HIV-1 antigen. Furthermore, sera from the modified HIV-1 antigen with defensins found to exhibit higher neutralizing activities (ranging from 59.3% to 84.6%) than the standard HIV-1 antigen. These results show that the induction of MPER-specific HIV-1 neutralizing antibodies could be achieved through a rationally designed vaccine strategy.

A synthetic chimeric peptide harboring human papillomavirus 16 cytotoxic T lymphocyte epitopes shows therapeutic potential in a murine model of cervical cancer.

Infection with human papillomavirus (HPV) such as HPV16 is known to be associated with cervical cancer. The E6 and E7 oncoproteins of this virus are attractive targets for T-cell-based immunotherapy to cervical cancer. In our study, software predicted, multiple H-2D(b) restricted HPV16 cytotoxic T lymphocytes (CTL) epitopes on a synthetic chimeric peptide, was used along with different immunopotentiating adjuvants such as alum, heat-killed Mycobacterium w (Mw) cells, and poly D,L-lactic-co-glycolide (PLGA) microspheres. We have shown that subcutaneous immunization with H-2D(b)-restricted HPV16 peptide was able to generate CTL-mediated cytolysis of HPV16 E6- and E7-expressing TC-1 tumor cells in vitro, as well as protect against in vivo challenge with TC-1 cells in C57BL/6 mice. In vitro, this chimeric peptide showed best efficacy with PLGA microspheres, moderate with alum, and least with Mw as adjuvant. This approach may thus provide a potential peptide-based therapeutic candidate vaccine for the control of HPV infection and hence cervical cancer.

Modulation of HIV peptide antigen specific cellular immune response by synthetic α- and ß-defensin peptides.

Defensin peptides have their direct role in host defense against microbial infection as innate molecules and also thought to contribute to adaptive immunity by recruiting naïve T-cells and immature dendritic cells at the site of infection through CCR6 receptor. The main aim of the present study is to investigate the efficacy of defensins for the induction of cell mediated immune response against the peptide antigen of HIV-1 encapsulated in PLG microparticles through intranasal (IN) route in mice model. To characterized, we have analyzed T-cell proliferation, Th1/Th2 cytokines, ß-chemokines production and IFN-γ/perforin secretion from CD4(+)/CD8(+) T-cells in response to HIV immunogen alone and with defensins at different mucosal site i.e. lamina propria (LP), spleen (SP) and peyer's patches (PP). The cellular immunogenicity of HIV peptide with defensin formulations showed a significantly higher (p<0.001) proliferation response as compared to individual HIV peptide. The enhanced cytokines measurement profile showed mixed Th1 and Th2 type of peptide specific immune response by the incorporation of defensins. In the continuation, enhancement in MIP-1α and RANTES level was also observed in HIV peptide-defensin formulations. The FACS data had revealed that CD4(+)/CD8(+) T-cells showed significantly (p<0.001) higher IFN-γ and perforin secretion in HIV with defensin peptide formulations than HIV antigen alone group. Thus, the study emphasized here that defensin peptides have a potential role as mucosal adjuvant, might be responsible for the induction of cell mediated immunity when administered in mice through IN route with HIV peptide antigen.

Novel adjuvants & delivery vehicles for vaccines development: a road ahead.

The pure recombinant and synthetic antigens used in modern day vaccines are generally less immunogenic than older style live/attenuated and killed whole organism vaccines. One can improve the quality of vaccine production by incorporating immunomodulators or adjuvants with modified delivery vehicles viz. liposomes, immune stimulating complexes (ISCOMs), micro/nanospheres apart from alum, being used as gold standard. Adjuvants are used to augment the effect of a vaccine by stimulating the immune system to respond to the vaccine, more vigorously, and thus providing increased immunity to a particular disease. Adjuvants accomplish this task by mimicking specific sets of evolutionary conserved molecules which include lipopolysaccharides (LPS), components of bacterial cell wall, endocytosed nucleic acids such as dsRNA, ssDNA and unmethylated CpG dinucleotide containing DNA. This review provides information on various vaccine adjuvants and delivery vehicles being developed to date. From literature, it seems that the humoral immune responses have been observed for most adjuvants and delivery platforms while viral-vector, ISCOMs and Montanides have shown cytotoxic T-cell response in the clinical trials. MF59 and MPL® have elicited Th1 responses, and virus-like particles (VLPs), non-degradable nanoparticle and liposomes have also generated cellular immunity. Such vaccine components have also been evaluated for alternative routes of administration with clinical success reported for intranasal delivery of viral-vectors and proteosomes and oral delivery of VLP vaccines.