Induction Of Cellular Immune Responses Research Articles

Silicon or Calcium Doping Coordinates the Immunostimulatory Effects of Aluminum Oxyhydroxide Nanoadjuvants in Prophylactic Vaccines.

Aluminum salts still remain as the most popular adjuvants in marketed human prophylactic vaccines due to their capability to trigger humoral immune responses with a good safety record. However, insufficient induction of cellular immune responses limits their further applications. In this study, we prepare a library of silicon (Si)- or calcium (Ca)-doped aluminum oxyhydroxide (AlOOH) nanoadjuvants. They exhibit well-controlled physicochemical properties, and the dopants are homogeneously distributed in nanoadjuvants. By using Hepatitis B surface antigen (HBsAg) as the model antigen, doped AlOOH nanoadjuvants mediate higher antigen uptake and promote lysosome escape of HBsAg through lysosomal rupture induced by the dissolution of the dopant in the lysosomes in bone marrow-derived dendritic cells (BMDCs). Additionally, doped nanoadjuvants trigger higher antigen accumulation and immune cell activation in draining lymph nodes. In HBsAg and varicella-zoster virus glycoprotein E (gE) vaccination models, doped nanoadjuvants induce high IgG titer, activations of CD4+ and CD8+ T cells, cytotoxic T lymphocytes, and generations of effector memory T cells. Doping of aluminum salt-based adjuvants with biological safety profiles and immunostimulating capability is a potential strategy to mediate robust humoral and cellular immunity. It potentiates the applications of engineered adjuvants in the development of vaccines with coordinated immune responses.

Pentavalent outer membrane vesicles immunized mice sera confers passive protection against five prevalent pathotypes of diarrhoeagenic Escherichia coli in neonatal mice

Diarrhoeagenic Escherichia coli (DEC) pathotypes are one of the major causative agents of diarrhoea induced childhood morbidity and mortality in developing countries. Licensed vaccines providing broad spectrum protection against DEC mediated infections are not available. Outer membrane vesicles (OMVs) are microvesicles released by gram-negative bacteria during the growth phase and contain multiple immunogenic proteins. Based on prevalence of infections, we have formulated a pentavalent outer-membrane vesicles (POMVs) based immunogen targeting five main pathotypes of DEC responsible for diarrhoeal diseases. Following isolation, OMVs from five DEC pathotypes were mixed in equal proportions to formulate POMVs and 10 µg of the immunogen was intraperitoneally administered to adult BALB/c mice. Three doses of POMVs induced significant humoral immune response against whole cell lysates (WCLs), outer membrane proteins (OMPs) and lipopolysaccharides (LPS) isolated from DEC pathotypes along with significant induction of cellular immune response in adult mice. Passive transfer of POMVs immunized adult mice sera protected neonatal mice significantly against DEC infections. Overall, this study finds POMVs to be immunogenic in conferring broad-spectrum passive protection to neonatal mice against five main DEC pathotypes. Altogether, these findings suggest that POMVs can be used as a potent vaccine candidate to ameliorate the DEC-mediated health burden.

Preclinical proof of concept of a tetravalent lentiviral T-cell vaccine against dengue viruses.

Dengue virus (DENV) is responsible for approximately 100 million cases of dengue fever annually, including severe forms such as hemorrhagic dengue and dengue shock syndrome. Despite intensive vaccine research and development spanning several decades, a universally accepted and approved vaccine against dengue fever has not yet been developed. The major challenge associated with the development of such a vaccine is that it should induce simultaneous and equal protection against the four DENV serotypes, because past infection with one serotype may greatly increase the severity of secondary infection with a distinct serotype, a phenomenon known as antibody-dependent enhancement (ADE). Using a lentiviral vector platform that is particularly suitable for the induction of cellular immune responses, we designed a tetravalent T-cell vaccine candidate against DENV ("LV-DEN"). This vaccine candidate has a strong CD8+ T-cell immunogenicity against the targeted non-structural DENV proteins, without inducing antibody response against surface antigens. Evaluation of its protective potential in the preclinical flavivirus infection model, i.e., mice knockout for the receptor to the type I IFN, demonstrated its significant protective effect against four distinct DENV serotypes, based on reduced weight loss, viremia, and viral loads in peripheral organs of the challenged mice. These results provide proof of concept for the use of lentiviral vectors for the development of efficient polyvalent T-cell vaccine candidates against all DENV serotypes.

Immunohistochemical and pathological changes in BALB/c mice immunized with whole sonicated Listeria monocytogenes antigens and the effect of probiotics

The current study was undertaken to investigate the role of macrophages as a cellular immune function against immunization with whole sonicated Listeria monocytogenes antigens (WSLMAgs) and the effect of probiotics. A preparation of WSLMAgs containing whole L. monocytogenes cell, after two subcutaneous immunization of BALB/c mice with 0.5ml WSLMAgs (0.5mg/ml) at an interval of two weeks. The bacterial identification was conducted by a conventional culture method using Listeria selective media PALCAM and confirmed by Polymerase Chain Reaction (PCR), As well as, the immunohistochemical and pathological change of it was studied in vivo by inoculating mice with pre-challenge WSLMAgs and post-challenge with virulent L. monocytogenes (1×108 CFU/mL). The results revealed the cellular immune function against pre- and post-immunization in spleen organ via lymphocytic hyperplasia in white pulp and coalescence of lymphoid follicles and marker F4/80+ show the immune-positive cells in aggregation adjacent to lymphoid follicle or focal aggregation of macrophages between follicles. In conclusion, the effectiveness of sonicated L. monocytogenes pre and post-immunization then challenge with virulent L. monocytogenes in the induction of cellular immune response, might serve as an immunization platform for applicants.

Evaluation of transduced dendritic cells expressing HIV-1 p24-Nef antigens in HIV-specific cytotoxic T cells induction as a therapeutic candidate vaccine

Various approaches have been investigated to prevent or eliminate HIV-1 since 1981. However, the virus has been affecting human population worldwide with no effective vaccine yet. The conserved regions among the viral genes are suitable targets in mutable viruses to induce the immune responses via an effective delivery platform.In this study, we aimed at evaluation of p24 and nef in two forms of full and truncated genes as two fusion antigenic forms according to our previous bioinformatics analysis. The designed antigens were then transferred through ex vivo generated dendritic cells and also proteins in BALB/c to assess and compare immunogenicity.p24 and Nef amino acid sequences were aligned, then, the most conserved regions were selected and two fusion forms as the truncated (p24:80–231aa-Nef:120–150aa) and the full from (p24-Nef) were cloned and expressed in prokaryotic and eukaryotic systems. Lentiviral vectors were applied to generate recombinant virions harboring the genes of interest to transduce generated murine dendritic cells. BALB/c mice received the recombinant DCs or recombinant proteins according to the defined schedule. IgG development was assessed to determine humoral immune activity and cellular immune responses were evaluated by IL-5 and IFN-y induction. Granzyme B secretion was also investigated to determine CTL activity in different immunized groups.The data showed high induction of cellular immune responses in dendritic cell immunization specifically in immunized mice with the truncated form of the p24 and Nef by high secretion of IFN-y and strong CTL activity. Moreover, protein/ DC prime-boost formulation led to stronger Th1 pathway and strong CTL activation in comparison with other formulations.The generated recombinant dendritic cells expressing p24-Nef induced humoral and cellular immunity in a Th1 pathway specifically with the in silico predicted truncated antigen which could be of high value as a dendritic cell therapeutic vaccine candidate against HIV-1.

AI Aided Design of Epitope-Based Vaccine for the Induction of Cellular Immune Responses Against SARS-CoV-2.

The heavy burden imposed by the COVID-19 pandemic on our society triggered the race toward the development of therapies or preventive strategies. Among these, antibodies and vaccines are particularly attractive because of their high specificity, low probability of drug-drug interaction, and potentially long-standing protective effects. While the threat at hand justifies the pace of research, the implementation of therapeutic strategies cannot be exempted from safety considerations. There are several potential adverse events reported after the vaccination or antibody therapy, but two are of utmost importance: antibody-dependent enhancement (ADE) and cytokine storm syndrome (CSS). On the other hand, the depletion or exhaustion of T-cells has been reported to be associated with worse prognosis in COVID-19 patients. This observation suggests a potential role of vaccines eliciting cellular immunity, which might simultaneously limit the risk of ADE and CSS. Such risk was proposed to be associated with FcR-induced activation of proinflammatory macrophages (M1) by Fu et al. (2020) and Iwasaki and Yang (2020). All aspects of the newly developed vaccine (including the route of administration, delivery system, and adjuvant selection) may affect its effectiveness and safety. In this work we use a novel in silico approach (based on AI and bioinformatics methods) developed to support the design of epitope-based vaccines. We evaluated the capabilities of our method for predicting the immunogenicity of epitopes. Next, the results of our approach were compared with other vaccine-design strategies reported in the literature. The risk of immuno-toxicity was also assessed. The analysis of epitope conservation among other Coronaviridae was carried out in order to facilitate the selection of peptides shared across different SARS-CoV-2 strains and which might be conserved in emerging zootic coronavirus strains. Finally, the potential applicability of the selected epitopes for the development of a vaccine eliciting cellular immunity for COVID-19 was discussed, highlighting the benefits and challenges of such an approach.

Live Vaccinia Virus-Coated Microneedle Array Patches for Smallpox Vaccination and Stockpiling.

Although smallpox has been eradicated globally, the potential use of the smallpox virus in bioterrorism indicates the importance of stockpiling smallpox vaccines. Considering the advantages of microneedle-based vaccination over conventional needle injections, in this study, we examined the feasibility of microneedle-based smallpox vaccination as an alternative approach for stockpiling smallpox vaccines. We prepared polylactic acid (PLA) microneedle array patches by micromolding and loaded a second-generation smallpox vaccine on the microneedle tips via dip coating. We evaluated the effect of excipients and drying conditions on vaccine stability in vitro and examined immune responses in female BALB/c mice by measuring neutralizing antibodies and interferon (IFN)-γ-secreting cells. Approximately 40% of the virus titer was reduced during the vaccine-coating process, with or without excipients. At −20 °C, the smallpox vaccine coated on the microneedles was stable up to 6 months. Compared to natural evaporation, vacuum drying was more efficient in improving the smallpox vaccine stability. Microneedle-based vaccination of the mice elicited neutralizing antibodies beginning 3 weeks after immunization; the levels were maintained for 12 weeks. It significantly increased IFN-γ-secreting cells 12 weeks after priming, indicating the induction of cellular immune responses. The smallpox-vaccine-coated microneedles could serve as an alternative delivery system for vaccination and stockpiling.

Lymphoid follicle antigen (Ag) delivery and enhanced rodent humoral immune responses mediated by Ag-containing PEGylated liposomes

Antigen (Ag) delivery to lymphoid follicles is important in achieving adaptive immunity. We recently developed a novel two-step Ag delivery system that efficiently induces cellular immune responses to Ags in mice by using priming intravenous (i.v.) injections of empty PEGylated liposomes (PEG-Lip) followed 3 days later by Ag-entrapped PEG-Lip (Ag-PEG-lip). In this study, we looked for humoral immune responses in rats and mice with IgG production specific to the encapsulated Ags. We observed that initial i.v. injections of empty PEG-Lip triggered accumulation of subsequent doses ovalbumin-PEG-Lip (OVA-PEG-lip) in splenic follicles and enhanced IgG production against OVA in both rats and mice. Anti-OVA IgG production was diminished by inhibition of splenic follicular accumulation of OVA-PEG-Lip by fingolimod (FTY720), which inhibits lymphocyte egress from lymphoid tissues. Thisindicates that the follicular accumulation of Ags that we observed is an indispensable and unique step in the production of anti-OVA IgG. Interestingly, in BALB/c nude mice, which are T cell deficient, a high follicular accumulation of OVA-PEG-Lip was observed, but anti-OVA IgG production was not observed. This suggests that T cells are also indispensable for the induction of cellular immune responses by our two-step immunization procedure. Our unique Ag delivery platform, which efficiently delivers Ags to splenic follicles, may be a useful technique for the enhancement of cellular immunity, as well as humoral immunity. Further experimental evaluation should be undertaken in relevant animal models in order for efficacy, safety and immunological correlates to be determined.

Selective Stimulation of Duplicated Atlantic Salmon MHC Pathway Genes by Interferon-Gamma.

Induction of cellular immune responses rely on Major histocompatibility complex (MHC) molecules presenting pathogenic peptides to T cells. Peptide processing, transport, loading and editing is a constitutive process in most cell types, but is accelerated upon infection. Recently, an unexpected complexity in the number of functional genes involved in MHC class I peptide cleavage, peptide transport, peptide loading and editing was found in teleosts, originating from the second and third whole genome duplication events. Salmonids have expanded upon this with functional duplicates also from a fourth unique salmonid whole genome duplication. However, little is known about how individual gene duplicates respond in the context of stimulation. Here we set out to investigate how interferon gamma (IFNg) regulates the transcription of immune genes in Atlantic salmon with particular focus on gene duplicates and MHC pathways. We identified a range of response patterns in Atlantic salmon gene duplicates, with upregulation of all duplicates for some genes, like interferon regulatory factor 1 (IRF1) and interferon induced protein 44-like (IFI44.L), but only induction of one or a few duplicates of other genes, such as TAPBP and ERAP2. A master regulator turned out to be the IRF1 and not the enhanceosome as seen in mammals. If IRF1 also collaborates with CIITA and possibly NLRC5 in regulating IFNg induction of MHCI and MHCII expression in Atlantic salmon, as in zebrafish, remains to be established. Altogether, our results show the importance of deciphering between gene duplicates, as they often respond very differently to stimulation and may have different biological functions.

Mechanistic insight into the induction of cellular immune responses by encapsulated and admixed archaeosome-based vaccine formulations

ABSTRACT Archaeosomes are liposomes formulated using total polar lipids (TPLs) or semi-synthetic glycolipids derived from archaea. Conventional archaeosomes with entrapped antigen exhibit robust adjuvant activity as demonstrated by increased antigen-specific humoral and cell-mediated responses and enhanced protective immunity in various murine infection and cancer models. However, antigen entrapment efficiency can vary greatly resulting in antigen loss during formulation and variable antigen:lipid ratios. In order to circumvent this, we recently developed an admixed archaeosome formulation composed of a single semi-synthetic archaeal lipid (SLA, sulfated lactosylarchaeol) which can induce similarly robust adjuvant activity as an encapsulated formulation. Herein, we evaluate and compare the mechanisms involved in the induction of early innate and antigen-specific responses by both admixed (Adm) and encapsulated (Enc) SLA archaeosomes. We demonstrate that both archaeosome formulations result in increased immune cell infiltration, enhanced antigen retention at injection site and increased antigen uptake by antigen-presenting cells and other immune cell types, including neutrophils and monocytes following intramuscular injection to mice using ovalbumin as a model antigen. In vitro studies demonstrate SLA in either formulation is preferentially taken up by macrophages. Although the encapsulated formulation was better able to induce antigen-specific CD8+ T cell activation by dendritic cells in vitro, both encapsulated and admixed formulations gave equivalently enhanced protection from tumor challenge when tested in vivo using a B16-OVA melanoma model. Despite some differences in the immunostimulatory profile relative to the SLA (Enc) formulation, SLA (Adm) induces strong in vivo immunogenicity and efficacy, while offering an ease of formulation.

Design and adjuvant formulation of a mutant HPV-E7 protein devoid of transforming/oncogenic properties but retaining high anti-tumor cellular activities as a candidate immunotherapeutic vaccine - Bioinformatics and in vivo analyses

Background & Aim Infection with Human papillomavirus (HPV) is major cause of the cervical carcinoma. Approved vaccines based on induction of antibodies (Abs) against HPV-L1 capsid protein provide prophylactic immunity against infection but no therapeutic effect on already infected individuals who might develop cancer. Alternatively, for anti-tumor immunotherapy purposes, induction of cellular immune responses against HPV oncogenic proteins, especially E7 is required. The oncogenic potential of HPV-E7 antigen (Ag) however, promoted us to search for a modified (mutated/deleted) form of this Ag with eliminated transforming properties but retained antigenicity. Methods, Results & Conclusion Herein, we first present application of several immune-informatics tools to evaluate the changes in the antigenicity, MHC binding/processing and cellular responses of the reported HPV16-E7 mutations responsible for its main transformation capabilities including del 21-24, C24G, L67R, C58G,C91G, M84S, V69A, L79A, QKP96-98EEA and their various combinations. Finally, the HPV16-E7 with triple substitutions of C24G/L67R/C91G (mE7) which showed the maximal antigenic similarity profile to that of the wild type was synthesized and inserted in pET28a vector for expression of protein in E.coli. SDS-PAGE and WB analyses confirmed the expression of the 17 kDa recombinant mE7 protein which was purified by NI-NTA chromatography using the vector encoded 6xHis-taq. The E.coli-derived Ag in combination with human compatible adjuvants including: Montanaide ISA 50 (M50), ISA 720 (M720), Pluronic acid (F127) in mixture with Immunostimulatory CpG ODN 1826 (mE7+M50/CpG; mE7+M720/CpG; mE7+F127/CpG, respectively) was evaluated in immunized C57BL/6 mice. Evaluation of the lymphocytes replication by the Brdu method and cytokine measurements by ELISA indicated that all groups developed significant immune responses, but mice immunized with mE7+M50/CpG showed the highest stimulation index (0.4) and INF-γ levels (150 pg/ml) comparable to those immunized by mE7+complete/incomplete Freund adjuvants (C/IFA). Accordingly, the IL-4 cytokine levels were almost similar and lower (20 pg/ml) for all groups indicating a Th1 polarization. Results of the tumor-challenge studies on TC1 cells in tumor bearing mice showed complete reduction/inhibition of tumor growth for mE7+M50/CpG immunized mice, indicating the potency of this formulation as a candidate immunotherapeutic vaccine against HPV infection-induced cancer.

Microfold cell-dependent antigen transport alleviates infectious colitis by inducing antigen-specific cellular immunity.

Infectious colitis is one of the most common health issues worldwide. Microfold (M) cells actively transport luminal antigens to gut-associated lymphoid tissue to induce IgA responses; however, it remains unknown whether M cells contribute to the induction of cellular immune responses. Here we report that M cell-dependent antigen transport plays a critical role in the induction of Th1, Th17, and Th22 responses against gut commensals in the steady state. The establishment of commensal-specific cellular immunity was a prerequisite for preventing bacterial dissemination during enteropathogenic Citrobacter rodentium infection. Therefore, M cell-null mice developed severe colitis with increased bacterial dissemination. This abnormality was associated with mucosal barrier dysfunction. These observations suggest that antigen transport by M cells may help maintain gut immune homeostasis by eliciting antigen-specific cellular immune responses.

A Mini Review on the Corona Various- A Universal Long Standing Problem: T-Cell Therapy Might Show Some Light in New Vaccine Development

Stimulation of the body’s immune system might be important to cure many diseases. The induction of cellular immune responses has thus far received little attention in respiratory virus vaccine development. CD8 T cells are critical for mediating clearance following many acute viral infections in the lung. In addition, memory CD8 T cells are also capable of providing protection against secondary infections. The present mini reviewed the present status of corona virus and was attempted to elucidate the future danger of corona virus. A suggestion for the development of T-cell based therapies has been made to fight against Corona virus. The enhancement of immunity using T-cell therapy might show a new light in this regard. Because of genetic diversity and frequent mutational change, it appears to be difficult to find a permanent solution of the corona problem in the near future. Vaccinations that solely promote the induction of neutralizing antibodies may not be optimal in providing protection against the corona virus respiratory infections, in particular. Therefore, human civilization is going to face unprecedented experiences, if not social and individual cares are always maintained and common people understand the seriousness of the virus.

Remote induction of cellular immune response in mice by anti-meningococcal nanocochleates - nanoproteoliposomes

New adjuvant formulations, based on proteoliposomes <40 nm and cochleates <100 nm, without Al(OH)3 adjuvant, were evaluated regarding their ability to generate Th1 immune response through a Delayed -Type Hypersensitivity Test, at the mouse model, by using a Neisseria meningitidis B protein complex as antigen. The formulations were administered by intramuscular (IM) (2 inoculations - at baseline and after 14 days) and intranasal (IN) (3 inoculations at 7 days) immunization pathways. All IM immunized groups were able to induce similar response to these formulations as well as to VA-MENGOC-BC® vaccine - containing Al(OH)3 adjuvant (used as positive control of the trial). In all groups, the induced inflammation (IP) rate was statistically higher than in the negative control group (CN) (p < 0.05). Immunogenicity, measured by HSR and CD4+ lymphocyte increase was equivalent to the control vaccine and most important, granuloma reactogenicity at the site of injection was eliminated, fact demonstrated by histological study. All groups of animals immunized by IN route showed HSR reactions and statistically significant differences with respect to the CN group. However, IP values were lower, with statistical differences (p < 0.05) for the same adjuvant formulation IM administered, except the AIF2-nCh formulation that generated statistically similar induction (p > 0.05) by both immunization pathways, suggesting it to be the best candidate for the next IN trial. Proteoliposome and cochleate formulations tested were able to mount potent Th-1 immune response, equivalent to the original vaccine formulation, with the advantage of less reactogenicity in the site of the injection, caused by the toxicity of Al(OH)3 adjuvant gel.

IL-33 Is a Negative Regulator of Vaccine-Induced Antigen-Specific Cellular Immunity.

The cytokine IL-33 is a well-established inducer of Th2 responses. However, roles for IL-33 in promoting CD8, Th1, and T regulatory cell responses have also emerged. In this study, the role of IL-33 as a regulator of particulate vaccine adjuvant-induced Ag-specific cellular immunity was investigated. We found that polymeric nanoparticles surpassed alum in their ability to enhance Ag-specific CD8 and Th1 responses. IL-33 was a potent negative regulator of both CD8+ T cell and Th1 responses following i.m. vaccination with Ag and nanoparticles, whereas the cytokine was required for the nanoparticle enhancement in Ag-specific IL-10. In contrast to the effect on cellular immunity, Ab responses were comparable between vaccinated wild-type and IL-33-deficient mice. IL-33 did not compromise alum-induced adaptive cellular immunity after i.m. vaccination. These data suggest that IL-33 attenuates the induction of cellular immune responses by nanoparticulate adjuvants and should be considered in the rational design of vaccines targeting enhanced CD8 and Th1 responses.

Formulation of HBs antigen in Montanide ISA266 shows superiority to commercial HBsAg vaccine in the induction of humoral immune responses.

In the present study, a new formulation of HBsAg vaccine was developed and compared with a commercial peer. Vaccination of hepatitis B infection has been an unavoidable affair since the 1980s, though it has numerous limitations such as inefficacy in the induction of cellular immune responses. To address these limitations, research on novel formulations is necessary to develop a superior formulation with the potency of induction of both cellular and humoral immune responses. HBsAg was formulated in oil-in-water adjuvant Montanide ISA-266 (5 µg/dose) using homogenizer. Balb/C mice were then immunized three times at days 0, 14, and 28 with HBsAg/Montanide ISA-266 or HBsAg/alum with proper control groups. Two weeks after the last immunization, immunological parameters including IL-2, IL-4, TNF-α, IFN-γ, total IgG and IgG1/IgG2a isotypes were assessed by ELISA. The results demonstrated that the formulation of HBsAg with Montanide ISA-266 enhanced humoral immune responses versus the commercial vaccine and control groups. No significant difference in terms of Th1 pattern was found between HBsAg/Montanide ISA-266 and the commercial vaccine. Formulation of HBsAg with an oil-based adjuvant may be useful for the induction of a more potent humoral immune response compared to the commercially available HBV vaccine.

In vivo electroporation enhances vaccine-mediated therapeutic control of human papilloma virus-associated tumors by the activation of multifunctional and effector memory CD8+ T cells

In vivo electroporation (EP) has reignited the clinical interest on DNA vaccines as immunotherapeutic approaches to control different types of cancer. EP has been associated with increased immune response potency, but its capacity in influencing immunomodulation remains unclear. Here we evaluated the impact of in vivo EP on the induction of cellular immune responses and therapeutic effects of a DNA vaccine targeting human papillomavirus-induced tumors. Our results demonstrate that association of EP with the conventional intramuscular administration route promoted a more efficient activation of multifunctional and effector memory CD8+ T cells with enhanced cytotoxic activity. Furthermore, EP increased tumor infiltration of CD8+ T cells and avoided tumor recurrences. Finally, our results demonstrated that EP promotes local migration of antigen presenting cells that enhances with vaccine co-delivery. Altogether the present evidences shed further light on the in vivo electroporation action and its impact on the immunogenicity of DNA vaccines.

Comparative Study of 5'- and 3'-Linked CpG-Antigen Conjugates for the Induction of Cellular Immune Responses.

Conjugation of CpG to an antigen induces a stronger immune response compared to that of the mixture. This study compares the in vitro immunostimulatory activity of CpG conjugated via either its 5′ or 3′ end to the model antigen ovalbumin (OVA). CpG modified with an amine at either the 5′ or 3′ end was conjugated to OVA via a stable bis-aryl hydrazone bond. Similar levels of CpG conjugation to OVA were observed for both conjugates on the basis of the absorbance at 360 nm for the formation of the bis-aryl hydrazone bond, which determined 2.8 ± 0.3 CpGs linked per OVA. Both the 5′ and 3′ CpG–OVA conjugates had similar size-exclusion chromatography elution profiles. The immunostimulatory properties of the conjugates were determined by dendritic cells (DCs) and T-cells isolated from mice. The activation of DCs was determined by the upregulation of activation markers CD86 and CD40. T-cells were co-cultured with stimulated DCs, and the immunogenicity was determined by measuring T-cell proliferation and interferon γ production. Both the CpG 5′- and 3′-linked conjugates induced the same level (p > 0.5) of DC activation markers, which were significantly higher than those of the untreated control. Similarly, T-cell assays showed no significant difference (p > 0.5) between the 5′ and 3′ conjugates with respect to T-cell proliferation and interferon γ production. The 5′ and 3′ conjugates induced T-cell activation significantly higher than the mixture of CpG and OVA. This study showed that the end at which CpG is conjugated to an antigen has no influence on the generation of a T-cell-based immune response in vitro.

Expression and Characterization of Yeast Derived Chikungunya Virus Like Particles (CHIK-VLPs) and Its Evaluation as a Potential Vaccine Candidate.

Chikungunya virus (CHIKV) has emerged as a global health concern due to its recent spread in both old and new world. So far, no CHIKV specific drug or vaccine is licensed for human use. In this study, we report production of Chikungunya virus like particles (CHIK-VLPs) using novel yeast expression system (Pichia pastoris) and its evaluation as vaccine candidate. The gene encoding structural polyprotein of CHIKV from a recent epidemic strain was cloned into yeast expression system. The multicopy integrants were processed for expression of CHIK-VLPs. The VLPs were purified and confirmed through electron microscopic analysis for their morphological identity with CHIKV. The in vitro and in vivo evaluation of CHIK-VLPs as vaccine candidate was determined in Balb/c mice. Induction of both humoral and cellular immune response was observed with different doses of CHIK-VLPs. The humoral immune response was studied through different techniques like enzyme linked immunosorbent assay, IgG Isotyping and plaque reduction neutralization test. CHIK-VLPs were found to elicit high titer of antibodies that are able to recognize native CHIKV. Higher level of IgG2a and IgG1 subtypes was identified suggestive of balanced Th1/Th2 response. Both in vitro and in vivo neutralization activity of CHIK-VLPs antibodies was observed even with low concentration, which shows its high specificity and neutralizing activity against two different CHIKV strains. Neonatal mice receiving anti-CHIK-VLPs antibodies were protected from CHIKV challenge. Induction of cellular immune response was confirmed through higher level of TNF-α, IL-10 and substantial level of IL-2, IL-4 and IFN-γ indicating a balanced response. This is the first report, where CHIK-VLPs has been expressed by Pichia pastoris and evaluated for neutralizing activity against CHIKV. These promising results indicate the utility of CHIK-VLPs as a promising vaccine candidate against emerging CHIKV.

Complement-Opsonized HIV-1 Overcomes Restriction in Dendritic Cells.

DCs express intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. Thus, DCs are productively infected only at very low levels with HIV-1, and this non-permissiveness of DCs is suggested to go along with viral evasion. We now illustrate that complement-opsonized HIV-1 (HIV-C) efficiently bypasses SAMHD1 restriction and productively infects DCs including BDCA-1 DCs. Efficient DC infection by HIV-C was also observed using single-cycle HIV-C, and correlated with a remarkable elevated SAMHD1 T592 phosphorylation but not SAMHD1 degradation. If SAMHD1 phosphorylation was blocked using a CDK2-inhibitor HIV-C-induced DC infection was also significantly abrogated. Additionally, we found a higher maturation and co-stimulatory potential, aberrant type I interferon expression and signaling as well as a stronger induction of cellular immune responses in HIV-C-treated DCs. Collectively, our data highlight a novel protective mechanism mediated by complement opsonization of HIV to effectively promote DC immune functions, which might be in the future exploited to tackle HIV infection.