Humoral Immune Responses In Mice Research Articles

A Lipid Nanoparticle-Formulated Self-Amplifying RNA Rift Valley Fever Vaccine Induces a Robust Humoral Immune Response in Mice

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis that causes high fetal and neonatal mortality rates in ruminants and sometimes severe to fatal complications like encephalitis and hemorrhagic fever in humans. There is no licensed RVF vaccine for human use while approved livestock vaccines have suboptimal safety or efficacy. We designed self-amplifying RNA (saRNA) RVF vaccines and assessed their humoral immunogenicity in mice. Plasmid DNA encoding the Rift Valley fever virus (RVFV) medium (M) segment consensus sequence (WT consensus) and its derivatives mutated to enhance cell membrane expression of the viral surface glycoproteins n (Gn) and c (Gc) were assessed for in vitro expression. The WT consensus and best-expressing derivative (furin-T2A) were cloned into a Venezuelan equine encephalitis virus (VEEV) plasmid DNA replicon and in vitro transcribed into saRNA. The saRNA was formulated in lipid nanoparticles and its humoral immunogenicity in BALB/c mice was assessed. High quantities of dose-dependent RVFV Gn IgG antibodies were detected in the serum of all mice immunized with either WT consensus or furin-T2A saRNA RVF vaccines. Significant RVFV pseudovirus-neutralizing activity was induced in mice immunized with 1 µg or 10 µg of the WT consensus saRNA vaccine. The WT consensus saRNA RVF vaccine warrants further development.

Immunogenicity of novel vB_EcoS_NBD2 bacteriophage-originated nanotubes as a carrier for peptide-based vaccines

Non-infectious virus-like nanoparticles mimic native virus structures and can be modified by inserting foreign protein fragments, making them immunogenic tools for antigen presentation. This study investigated, for the first time, the immunogenicity of long and flexible polytubes formed by yeast-expressed tail tube protein gp39 of bacteriophage vB_EcoS_NBD2 and evaluated their ability to elicit an immune response against the inserted protein fragments. Protein gp39-based polytubes induced humoral immune response in mice, even without the use of adjuvant. Bioinformatics analysis guided the selection of protein fragments from Acinetobacter baumannii for insertion into the C-terminus of gp39. Chimeric polytubes, displaying 28-amino acid long OmpA protein fragment, induced IgG response against OmpA protein fragment in immunized mice. These polytubes demonstrated their effectiveness both as antigen carrier and an adjuvant, when the OmpA fragments were either displayed on chimeric polytubes or used alongside with the unmodified polytubes. Our findings expand the potential applications of long and flexible polytubes, contributing to the development of novel antigen carriers with improved immunogenicity and antigen presentation capabilities.

Evaluation of the Immunity Responses in Mice to Recombinant Bacillus subtilis Displaying Newcastle Disease Virus HN Protein Truncations.

Bacillus subtilis, a probiotic bacterium with engineering potential, is widely used for the expression of exogenous proteins. In this study, we utilized the integrative plasmid pDG364 to integrate the hemagglutinin-neuraminidase (HN) gene from Newcastle disease virus (NDV) into the genome of the B. subtilis 168 model strain. We successfully constructed a recombinant B. subtilis strain (designated B. subtilis RH) that displays a truncated HN antigen fragment on the surface of its spores and further evaluated its immunogenic effects in mice. Using ELISA, we quantified the levels of IgG in serum and secretory IgA (sIgA) in intestinal contents. The results revealed that the recombinant B. subtilis RH elicited robust specific mucosal and humoral immune responses in mice. Furthermore, B. subtilis RH demonstrated potential mucosal immune adjuvant properties by fostering the development of immune organs and augmenting the number of lymphocytes in the small intestinal villi. Additionally, the strain significantly upregulated the relative expression of inflammatory cytokines such as IL-1β, IL-6, IL-10, TNF-α, and IFN-γ in the small intestinal mucosa. In conclusion, the B. subtilis RH strain developed in this study exhibits promising mucosal immunogenic effects. It holds potential as a candidate for an anti-NDV mucosal subunit vaccine and offers a novel preventive strategy for the poultry industry against this disease.

Construction and evaluation of a novel multi-antigenic Mycobacterium tuberculosis subunit vaccine candidate BfrB-GrpE/DPC

Tuberculosis poses a significant threat to human health due to the lack of an effective vaccine. Although promising progress has been made in the development of tuberculosis vaccines, new vaccines that broaden the antigenic repertoire need to be developed to eradicate this illness. In this study, we used Mycobacterium tuberculosis ferritin BfrB and heat-shock protein GrpE to construct a novel multi-antigenic fusion protein, BfrB-GrpE (BG). BG protein was stably overexpressed in the soluble form in Escherichia coli at a high yield and purified via sequential salt fractionation and hydrophobic chromatography. Purified BG was emulsified in an adjuvant containing N, N'-dimethyl-N, N'-dioctadecylammonium bromide, polyinosinic-polycytidylic acid, and cholesterol (DPC) to construct the BG/DPC vaccine, which stimulated strong cellular and humoral immune responses in mice. Moreover, combination of BG with our previously developed vaccine, Mtb10.4-HspX (MH), containing antigens from both the proliferating and dormant stages, significantly reduced the bacterial counts in the lungs and spleens of M. tuberculosis-infected mice. Importantly, mice that received BG + MH/DPC after M. tuberculosis H37Rv infection survived slightly better (100% survival) than those that received the BCG vaccine (80% survival), although the difference was not statistically significant. Our findings can aid in the selection of antigens and optimization of vaccination regimens to improve the efficacy of tuberculosis vaccines.

Humoral Immune Response of Mice against a Vaccine Candidate Composed of a Chimera of gB of Bovine Alphaherpesviruses 1 and 5.

Infectious bovine rhinotracheitis (IBR) and bovine meningoencephalitis are caused by Bovine alphaherpesvirus (BoHV) types 1 and 5, which seriously threaten the global cattle industry. Vaccination to improve immunity is the most direct and effective means to prevent these conditions. Glycoprotein B (gB) is essential for the attachment of both viruses to permissive cells, and is a major target of the host immune system, inducing a strong humoral response. The aim of this study was to evaluate, in a murine model, the immune response of a candidate vaccine formulation composed of a chimeric BoHV-1 and BoHV-5 gB (DgB), expressed in Komagataella phaffii. The chimeric DgB vaccine adjuvanted with Montanide 50 ISA V2 or aluminum hydroxide was administered intramuscularly or subcutaneously. A control group and a group that received a commercial vaccine were inoculated subcutaneously. Higher titers of neutralizing antibodies against BoHV-1, BoHV-5, and a natural BoHV-1/5 recombinant strain were obtained with the oil-based candidate vaccine formulation administered intramuscularly. The results demonstrated that the chimeric DgB conserved important epitopes that were able to stimulate a humoral immune response capable of neutralizing BoHV-1, BoHV-5, and the recombinant strain, suggesting that the vaccine antigen is a promising candidate to be further evaluated in cattle.

B602L-Fc fusion protein enhances the immunogenicity of the B602L protein of the African swine fever virus.

African swine fever (ASF) is an acute, highly contagious, and deadly infectious disease caused by the African swine fever virus (ASFV) and has a huge impact on the pig industry. A lack of vaccines and effective therapeutic drugs has brought great challenges to the prevention and control of ASF. In this study, insect baculovirus expression system was used to express ASFV B602L protein (B602L) alone and the IgG FC-fused B602L protein (B602L-Fc), and evaluate the immune effect of B602L-Fc in mice model. To be specific, the ASFV B602L protein and B602L-Fc fusion protein were successfully expressed by the insect baculovirus expression system. Then, Functional analysis in vitro revealed that the B602L-Fc fusion protein bound and interacted with the FcRI receptor of antigen-presenting cells and significantly promoted the expression of proteins involved in antigen presentation and various cytokines at mRNA levels in porcine alveolar macrophages. Additionally, immunization using B602L-Fc fusion protein remarkably promoted the Th1-biased cellular immune response and humoral immune response in mice. In conclusion, The B602L-Fc fusion protein could up-regulate the expression of molecules involved in antigen presentation in APCs and enhance the humoral and cellular immune responses in mice. These results suggest that ASFV B602L-Fc recombinant fusion protein may be a promising candidate for subunit vaccine. This study provided useful data for the development of subunit vaccines for ASF.

Co-transient expression of PSA-Fc and PAP-Fc fusion protein in plant as prostate cancer vaccine candidates and immune responses in mice.

PAP-FcK and PSA-FcK prostate cancer antigenic proteins transiently co-expressed in plant induce their specific humoral immune responses in mice. Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) have been considered as immunotherapeutic antigens for prostate cancer. The use of a single antigenic agent is unlikely to be effective in eliciting immunotherapeutic responses due to the heterogeneous and multifocal nature of prostate cancer. Thus, multiple antigens have been combined to enhance their anti-cancer effects. In the current study, PSA and PAP were fused to the crystallizable region (Fc region) of immunoglobulin G1 and tagged with KDEL, the endoplasmic reticulum (ER) retention signal motif, to generate PSA-FcK and PAP-FcK, respectively, and were transiently co-expressed in Nicotiana benthamiana. Western blot analysis confirmed the co-expression of PSA-FcK and PAP-FcK (PSA-FcK + PAP-FcK) with a 1:3 ratios in the co-infiltrated plants. PSA-FcK, PAP-FcK, and PSA-FcK + PAP-FcK proteins were successfully purified from N. benthamiana by protein A affinity chromatography. ELISA showed that anti-PAP and anti-PSA antibodies successfully detected PAP-FcK and PSA-FcK, respectively, and both detected PSA-FcK + PAP-FcK. Surface plasmon resonance (SPR) analysis confirmed the binding affinity of the plant-derived Fc fusion proteins to FcγRI/CD64. Furthermore, we also confirmed that mice injected with PSA-FcK + PAP-FcK produced both PSA- and PAP-specific IgGs, demonstrating their immunogenicity. This study suggested that the transient plant expression system can be applied to produce the dual-antigen Fc fusion protein (PSA-FcK + PAP-FcK) for prostate cancer immunotherapy.

Mucosal immunization with lipopeptides derived from conserved regions of SARS-CoV-2 antigens induce robust cellular and cross-variant humoral immune responses in mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has infected >600 million people in the ongoing global pandemic. Several variants of the SARS-CoV-2 have emerged in the last >2 years, challenging the continued efficacy of current COVID vaccines. Therefore, there is a crucial need to investigate a highly cross-protective vaccine effective against variants of SARS-CoV-2. In this study, we examined seven lipopeptides derived from highly conserved, immunodominant epitopes from the S, N, and M proteins of SARS-CoV-2, that are predicted to contain epitopes for clinically protective B cells, helper T cells (TH) and cytotoxic T cells (CTL). Intranasal immunization of mice with most of the lipopeptides led to significantly higher splenocyte proliferation and cytokine production, mucosal and systemic antibody responses, and induction of effector B and T lymphocytes in both lungs and spleen, compared to immunizations with the corresponding peptides without lipid. Immunizations with Spike-derived lipopeptides led to cross-reactive IgG, IgM and IgA responses against Alpha, Beta, Delta, and Omicron Spike proteins as well as neutralizing antibodies. These studies support their potential for development as components of a cross-protective SARS-CoV-2 vaccine.

DLin-MC3-Containing mRNA Lipid Nanoparticles Induce an Antibody Th2-Biased Immune Response Polarization in a Delivery Route-Dependent Manner in Mice.

mRNA-based vaccines have made a leap forward since the SARS-CoV-2 pandemic and are currently used to develop anti-infectious therapies. If the selection of a delivery system and an optimized mRNA sequence are two key factors to reach in vivo efficacy, the optimal administration route for those vaccines remains unclear. We investigated the influence of lipid components and immunization route regarding the intensity and quality of humoral immune responses in mice. The immunogenicity of HIV-p55Gag encoded mRNA encapsulated into D-Lin-MC3-DMA or GenVoy-ionizable lipid-based LNPs was compared after intramuscular or subcutaneous routes. Three sequential mRNA vaccines were administrated followed by a heterologous boost composed of p24-HIV protein antigen. Despite equivalent IgG kinetic profiles of general humoral responses, IgG1/IgG2a ratio analysis showed a Th2/Th1 balance toward a Th1-biased cellular immune response when both LNPs were administrated via the intramuscular route. Surprisingly, a Th2-biased antibody immunity was observed when DLin-containing vaccine was injected subcutaneously. A protein-based vaccine boost appeared to reverse this balance to a cellular-biased response correlated to an increase in antibody avidity. Our finding suggests that the intrinsic adjuvant effect of ionizable lipids appears to be dependent on the delivery route used, which could be relevant to reach potent and long-lasting immunity after mRNA-based immunization.

A vaccine delivery system promotes strong immune responses against SARS-CoV-2 variants.

Global coronavirus disease 2019(COVID-19) pandemics highlight the need of developing vaccines with universal and durable protection against emerging SARS-CoV-2 variants. Here we developed an extended-release vaccine delivery system (GP-diABZI-RBD), consisting the original SARS-CoV-2 WA1 strain receptor-binding domain (RBD) as the antigen and diABZI stimulator of interferon genes(STING) agonist in conjunction with yeast β-glucan particles (GP-diABZI) as the platform. GP-diABZI-RBD could activate STING pathway and inhibit SARS-CoV-2 replication. Compared to diABZI-RBD, intraperitoneal injection of GP-diABZI-RBD elicited robust cellular and humoral immune responses in mice. Using SARS-CoV-2 GFP/ΔN transcription and replication-competent virus-like particle system (trVLP), we demonstrated that GP-diABZI-RBD-prototype vaccine exhibited the strongest and durable humoral immune responses and antiviral protection; whereas GP-diABZI-RBD-Omicron displayed minimum neutralization responses against trVLP. By using pseudotype virus (PsVs) neutralization assay, we found that GP-diABZI-RBD-Prototype, GP-diABZI-RBD-Delta, and GP-diABZI-RBD-Gamma immunized mice sera could efficiently neutralize Delta and Gamma PsVs, but had weak protection against Omicron PsVs. In contrast, GP-diABZI-RBD-Omicron immunized mice sera displayed the strongest neutralization response to Omicron PsVs. Taken together, the results suggest that GP-diABZI can serve as a promising vaccine delivery system for enhancing durable humoral and cellular immunity against broad SARS-CoV-2 variants. Our study provides important scientific basis for developing SARS-CoV-2 VOC-specific vaccines.

Genetically engineered bacterial‐like particles induced specific cellular and humoral immunity as effective tick‐borne encephalitis virus vaccine

AbstractTick‐borne encephalitis (TBE) is a natural focal disease with fatal encephalitis induced by tick‐borne encephalitis virus (TBEV), seriously threatening human and public health. Protection of TBE depends on vaccination with inactivated vaccine, which requires high cost and multiple immunizations. Here, we construct genetically engineered bacterial‐like particles (BLPs) as an effective TBEV vaccine with simplified immunizations and improved immune efficacy. The TBEV BLPs involve the combination of the gram‐positive enhancer matrix from Lactococcus lactis, and TBEV envelope (E) protein expressed by genetically engineered recombinant baculovirus. The prepared TBEV BLPs can effectively stimulate the activation of dendritic cells to present the TBEV E proteins to T and B cells, leading to strong and durable cellular and humoral immune responses in mice. Surprisingly, the serum levels of specific IgG antibodies in mice remain about 106 at 6 months after the secondary immunization. Overall, the TBEV BLPs can be used as a potent vaccine candidate, laying the foundation for developing novel TBEV genetically engineered vaccines.

Immuno-modulatory Effects of Inactivated Dietzia Maris on the Selected Aspects of Cellular and Humoral Immune Responses in Mice.

The current study is an attempt to register the alterations in the immunological and histological parameters in mice arising from the administration of Dietza maris (D. maris) in order to confirm its protective properties. Mice underwent 7 days of treatment with three doses of D. maris. Then, animals were scrutinized in terms of body weight, relative weight of organs, delayed type of hypersensitivity (DTH) response, and hemagglutination titer (HT). The determination of villus height, villus width, crypt depth, villus/crypt ratio (V/C), Goblet cells, and intestinal epithelial lymphocyte (IEL) density in villi was carried out. A boosted DTH response was observed as a result of bacteria at medium dose. A variation was noted between the hemagglutinin titer of the control group and that of the high-dose group. Crypt depth, villus width, and villus height manifested alterations. High-dose-treated mice demonstrated proliferation of Goblet cells in the villi, whereas both in medium- and high-dose-treated mice, a distribution of IELs in the villus epithelium was noted. Overall, D. maris showed a stimulatory effect on immune functions in mice. Thus, thanks to improved cellular and humoral immunity and the increased quality of intestine function, we believe that D. maris promises novel therapeutic applications in the future. The attained findings lend credence to immuno-stimulatory effects arising from the capacity of D. maris to function as immunological adjuvants and to enhance humoral and cellular immunity as well as the intestinal structure and function.

Immunomosuppressive Effect of Ziziphus Lotus L. (Desf.) Fruit’s Extract on Neutrophil Bactericidal Activity and on in Vivo Humoral Immune Response in Mice.

Background: The fruit of Ziziphus Lotus L. (ZL) is rich in bioactive components. It is known for its high content in polyphenols which gives it its immunomodulatory, antioxidant, and antimicrobial properties. Objective: The intent of the current study was to evaluate, in vivo, the effect of the aqueous extract of ZL fruit’s pulp on humoral immune response as well as its effect on neutrophils’ bactericidal activities, hemolytic and antioxidant and activities. Methods: The antioxidant activity of ZL’s aqueous extract’s was evaluated using DPPH. Hemmagglutination titer assay was used to evaluate the effect of the extract on humoral immune response. ZL extract’s hemolytic activity was assessed by enumerating hemoglobin rates. The effect of ZL extract on the bactericidal activity of Neutrophils was evaluated using MTT colorimetric assay. Results / Discussion: A significant (P<0.05) immunosuppressive effect on humoral immunity (6-fold) was observed. Significant suppression (P<0.05) of the bactericidal activity of neutrophils treated with 0.5 and 1 g/ml of the extract was observed compared to untreated neutrophils. The extract exhibited a high antioxidant activity determined by DPPH test with an IC50 value 10-fold higher (P<0.05) than the IC50 of ascorbic acid. The highest hemolytic activity was found with the lowest concentration of the extract while the higher concentrations tested seem to have an anti-hemolytic activity with a dose dependent effect. Conclusion: The aqueous extract of ZL’s fruit pulp possesses an immunosuppressive activity on both the innate and adaptive immunity responses. Our results demonstrate an anti-oxidative activity as well as an ability to decrease neutrophil bactericidal hemolytic activities as well as humoral immune responses.

Small ruminant lentivirus capsid protein (SRLV-p25) antigenic structural prediction and immunogenicity to recombinant SRLV-rp25-coupled to immunostimulatory complexes based on glycyrrhizinic acid.

Small ruminant lentiviruses (SRLV) infect sheep and goats resulting in significant economic losses. This study evaluated for the first time the predicted conformational structure of the SRLV-capsid-protein 25 (SRLV-p25) and analyzed the antigenicity of recombinant protein (SRLV-rp25) in mice by coupling to an immunostimulatory complexes based on glycyrrhizinic acid liposomes (GAL) and tested plasma from goats and sheep naturally infected. Analysis in silico and conformational structure of SRLV-p25 (genotype B-FESC-752) showed similar characteristics to other lentiviral capsids. The efficient expression of SRLV-rp25 was confirmed by Western blot. The humoral immune responses in mice showed an increased level of antibodies from day 21 to 35 of the SRLV-rp25-GAL and SRLV-rp25-ISCOM® groups and the cellular immune response showed no significant difference in IL-10 levels (P>.05), however, a significant difference (P<.001) was observed when comparing SRLV-rp25-GAL with SRLV-rp25 groups. Immunoreactivity toward SRLV-rp25 revealed 61% of positive samples from naturally infected goats and sheep.

Chitosan particle stabilized Pickering emulsion/interleukin-12 adjuvant system for Pgp3 subunit vaccine elicits immune protection against genital chlamydial infection in mice.

Considering the shortcomings in current chlamydia infection control strategies, a major challenge in curtailing infection is the implementation of an effective vaccine. The immune response induced by C. trachomatis plasmid encoded Pgp3 was insufficient against C. trachomatis infection, which requires adjuvant applications to achieve the robust immune response induced by Pgp3. There is increasing promising in developing adjuvant systems relying on the delivery potential of Pickering emulsions and the immunomodulatory effects of interleukin (IL)-12. Here, owing to the polycationic nature, chitosan particles tended to absorb on the oil/water interphase to prepare the optimized chitosan particle-stabilized Pickering emulsion (CSPE), which was designed as a delivery system for Pgp3 protein and IL-12. Our results showed that the average droplets size of CSPE was 789.47 ± 44.26 nm after a series of optimizations and about 90% antigens may be absorbed by CSPE owing to the positively charged surface (33.2 ± 3mV), and CSPE promoted FITC-BSA proteins uptake by macrophages. Furthermore, as demonstrated by Pgp3-specific antibody production and cytokine secretion, CSPE/IL-12 system enhanced significantly higher levels of Pgp3-specific IgG, IgG1, IgG2a, sIgA and significant cytokines secretion of IFN-γ, IL-2, TNF-α, IL-4. Similarly, vaginal chlamydial shedding and hydrosalpinx pathologies were markedly reduced in mice immunized with Pgp3/CSPE/IL-12. Collectively, vaccination with Pgp3/CSPE/IL-12 regimen elicited robust cellular and humoral immune response in mice resulting in an obvious reduction of live chlamydia load in the vaginal and inflammatory pathologies in the oviduct, which further propells the development of vaccines against C. trachomatis infection.

Immune Responses to Plant-Derived Recombinant Colorectal Cancer Glycoprotein EpCAM-FcK Fusion Protein in Mice.

Epidermal cell adhesion molecule (EpCAM) is a tumor-associated antigen (TAA), which has been considered as a cancer vaccine candidate. The EpCAM protein fused to the fragment crystallizable region of immunoglobulin G (IgG) tagged with KDEL endoplasmic reticulum (ER) retention signal (EpCAM-FcK) has been successfully expressed in transgenic tobacco (Nicotiana tabacum cv. Xanthi) and purified from the plant leaf. In this study, we investigated the ability of the plant-derived EpCAM-FcK (EpCAM-FcKP) to elicit an immune response in vivo. The animal group injected with the EpCAM-FcKP showed a higher differentiated germinal center (GC) B cell population (~9%) compared with the animal group injected with the recombinant rhEpCAM-Fc chimera (EpCAM-FcM). The animal group injected with EpCAM-FcKP (~42%) had more differentiated T follicular helper cells (Tfh) than the animal group injected with EpCAM-FcM (~7%). This study demonstrated that the plant-derived EpCAM-FcK fusion antigenic protein induced a humoral immune response in mice.

Limited cross-variant immunity from SARS-CoV-2 Omicron without vaccination

SARS-CoV-2 Delta and Omicron are globally relevant variants of concern. Although individuals infected with Delta are at risk of developing severe lung disease, infection with Omicron often causes milder symptoms, especially in vaccinated individuals1,2. The question arises of whether widespread Omicron infections could lead to future cross-variant protection, accelerating the end of the pandemic. Here we show that without vaccination, infection with Omicron induces a limited humoral immune response in mice and humans. Sera from mice overexpressing the human ACE2 receptor and infected with Omicron neutralize only Omicron, but not other variants of concern, whereas broader cross-variant neutralization was observed after WA1 and Delta infections. Unlike WA1 and Delta, Omicron replicates to low levels in the lungs and brains of infected animals, leading to mild disease with reduced expression of pro-inflammatory cytokines and diminished activation of lung-resident T cells. Sera from individuals who were unvaccinated and infected with Omicron show the same limited neutralization of only Omicron itself. By contrast, Omicron breakthrough infections induce overall higher neutralization titres against all variants of concern. Our results demonstrate that Omicron infection enhances pre-existing immunity elicited by vaccines but, on its own, may not confer broad protection against non-Omicron variants in unvaccinated individuals.

A Bacterially Expressed SARS-CoV-2 Receptor Binding Domain Fused With Cross-Reacting Material 197 A-Domain Elicits High Level of Neutralizing Antibodies in Mice.

The Coronavirus disease 2019 (COVID-19) pandemic presents an unprecedented public health crisis worldwide. Although several vaccines are available, the global supply of vaccines, particularly within developing countries, is inadequate, and this necessitates a need for the development of less expensive, accessible vaccine options. To this end, here, we used the Escherichia coli expression system to produce a recombinant fusion protein comprising the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; residues 319–541) and the fragment A domain of Cross-Reacting Material 197 (CRM197); hereafter, CRMA-RBD. We show that this CRMA-RBD fusion protein has excellent physicochemical properties and strong reactivity with COVID-19 convalescent sera and representative neutralizing antibodies (nAbs). Furthermore, compared with the use of a traditional aluminum adjuvant, we find that combining the CRMA-RBD protein with a nitrogen bisphosphonate-modified zinc-aluminum hybrid adjuvant (FH-002C-Ac) leads to stronger humoral immune responses in mice, with 4-log neutralizing antibody titers. Overall, our study highlights the value of this E. coli-expressed fusion protein as an alternative vaccine candidate strategy against COVID-19.

Xenogeneic Humoral Immune Responses to Human Mesenchymal Stem Cells in Mice

Background and ObjectivesMany preclinical studies have been conducted using animal disease models to determine the effectiveness of human mesenchymal stem cells (hMSCs) for treating immune and inflammatory diseases based on the belief that hMSCs are not immunogenic across species. However, several researchers have suggested xenogeneic immune responses to hMSCs in animals, still without detailed features. This study aimed to investigate a xenogeneic humoral immune response to hMSCs in mice in detail.Methods and ResultsBalb/c mice were intraperitoneally injected with adipose tissue-derived or Wharton’s jelly-derived hMSCs. Sera from these mice were titrated for each isotype. To confirm specificity of the antibodies, hMSCs were stained with the sera and subjected to a flow cytometic analysis. Spleens were immunostained for proliferating cell nuclear antigen to verify the germinal center formation. Additionally, splenocytes were subjected to a flow cytometric analysis for surface markers including GL-7, B220, CD4, CD8, CD44, and CD62L. Similar experiments were repeated in C57BL/6 mice. The results showed increased IgG1 and IgG2a titers in the sera from Balb/c mice injected with hMSCs, and the titers were much higher in the secondary sera than in the primary sera. These antibodies were specifically stained the hMSCs. Germinal centers were observed in the spleen, and flow cytometric analysis of the splenocytes showed higher frequencies of centroblasts (B220＋ GL7＋) and memory T cells (CD62L＋ CD44＋) both in CD4＋ and CD8＋ subsets. Similar results were obtained for C57BL/6 mice.ConclusionshMSCs induced a humoral immune response in mice, with characters of T cell-dependent immunity.

Immunomodulatory Activity of Aqueous Extract of Polyherbal Formulation on Macrophage Function and Humoral Immune Response against SRBCs in Mice

Several herbal formulation were not properly documented due to poor scientific data as well as poor standard regulation in preparation and marketing. The study evaluate the effect of aqueous extract of Polyherbal formulation on Macrophages’ phagocytic function and Humoral immune response in Mice A total of Sixteen 16 Mice was used. Group I received normal saline, Group II-IV received 500 mg, 1000 mg, and 1500 mg of Polyherbal formulation respectively for 21 days each. The animals were sensitized and challenged with Sheep red blood cells at day 14th and 19th of the treatment respectively. On day 21st all the animals were injected with 0.1 ml Indian ink for carbon clearance assay and blood sample was collected at 1 minute and 15 minutes of the injection. The phagocytic function of Macrophages and humoral immune response were determined spectrophotometrically and Hemagglutination assay respectively. The results show that the highest carbonic particle clearances index (K) median score (Median=0.0228), Macrophage phagocytose index (α) median score (Median=3.249), organ weight index (g/100g) mean score (M=0.06633) and Hemagglutination antibody titre median score (Median=32.00) was recorded by Group IV when compared with other groups. The carbonic particle clearance (K) (p=0.02), phagocytic index of Macrophage (α) (p=0.03), and organ weight index (g/100g) (p&lt;0.0001) significantly increase with increase in the Polyherbal formulation concentration. Hemagglutination antibody titre against Sheep red blood cells (p=0.02) also significantly increased. The extract has potential immunostimulatory activity on both Macrophages’ phagocytic function and humoral immune response in mice thus could be useful in improving immune responses.