Strong Cellular Immune Responses Research Articles

Dynamic response landscape of immune cells identified immune dysfunction which predicts disease progression in COVID-19 infected patients.

During the development of COVID-19 caused by SARS-CoV-2 infection from mild disease to severe disease, it can trigger a series of complications and stimulate a strong cellular and humoral immune response. However, the precise identification of blood immune cell response dynamics and the relevance to disease progression in COVID-19 patients remains unclear. We propose for the first time to use changes in cell numbers to establish new subgroups, which were divided into four groups: first from high to low cell number (H_L_Group), first from low to high (L_H_Group), continuously high (H_Group), and continuously low (L_Group). It was found that in the course of disease development. In the T cell subgroup, the immune response is mainly concentrated in the H_L_Group cell type, and the complications are mainly in the L_H_Group cell type. In the NK cell subgroup, the moderate patients are mainly related to cellular immunity, and the severe patients are mainly caused by the disease, while severe patients are mainly related to complications caused by diseases. Our study provides a dynamic response of immune cells in human blood during SARS-CoV-2 infection and the first subgroup analysis using dynamic changes in cell numbers, providing a new reference for clinical treatment of COVID-19.

Protection against virulent Brucella spp. by gamma-irradiated B. ovis in BALB/c mice model.

PurposeBrucella spp. is a zoonosis that causes undulant fever in humans and abortion in livestock worldwide. Lately, it was conveyed that vaccines developed by irradiation have induced a strong cellular and humoral immune response which have made these types of vaccines highly effective.Materials and MethodsIn this study, we aimed to use the gamma-irradiated B. ovis as a vaccine and to study the humoral immune response and cytokines production in order to evaluate it for protecting mice against B. abortus 544, B. melitensis 16M, and B. ovis.ResultsThe humoral immune response in immunized mice with gamma-irradiated B. ovis showed a lasting for 8 weeks after immunization. Moreover, immunoglobulin G (IgG), IgG1, IgG2a, and IgG2b isotypes antibodies against B. ovis were observed after 4 and 8 weeks of the last immunization. It was noticed that the production of tumor necrosis factor-α, interferon-γ, and interleukin (IL)-10 continued after 4 and 8 weeks by splenocytes from immunized BALB/c mice, while no production of IL-4 or IL-5 was observed.ConclusionOur results indicate that the protection of BALB/c mice against B. melitensis 16M, B. abortus 544, and B. ovis was induced and the developed vaccine at our laboratory could stimulate similar protection to those induced by the traditional vaccine.

Enhancing Immune Responses to a DNA Vaccine Encoding Toxoplasma gondii GRA7 Using Calcium Phosphate Nanoparticles as an Adjuvant.

Toxoplasma gondii infects almost all warm-blooded animals, including humans. DNA vaccines are an effective strategy against T. gondii infection, but these vaccines have often been poorly immunogenic due to the poor distribution of plasmids or degradation by lysosomes. It is necessary to evaluate the antigen delivery system for optimal vaccination strategy. Nanoparticles (NPs) have been shown to modulate and enhance the cellular humoral immune response. Here, we studied the immunological properties of calcium phosphate nanoparticles (CaPNs) as nanoadjuvants to enhance the protective effect of T. gondii dense granule protein (GRA7). BALB/c mice were injected three times and then challenged with T. gondii RH strain tachyzoites. Mice vaccinated with GRA7-pEGFP-C2+nano-adjuvant (CaPNs) showed a strong cellular immune response, as monitored by elevated levels of anti-T. gondii-specific immunoglobulin G (IgG), a higher IgG2a-to-IgG1 ratio, elevated interleukin (IL)-12 and interferon (IFN)-γ production, and low IL-4 levels. We found that a significantly higher level of splenocyte proliferation was induced by GRA7-pEGFP-C2+nano-adjuvant (CaPNs) immunization, and a significantly prolonged survival time and decreased parasite burden were observed in vaccine-immunized mice. These data indicated that CaPN-based immunization with T. gondii GRA7 is a promising approach to improve vaccination.

Lentinan PLGA-stabilized pickering emulsion for the enhanced vaccination

Lentinan (LNT), a β-1,3-linked-d-glucan with β-1,6 glucose branches, is the main bioactive component extracted from Lentinus edodes. As a carbohydrate polymer, it has attracted increasingly attention because of immune enhancement effect. Pickering emulsion has been widely used in biomedicine due to its great stability, high loading capacity, and appreciable biocompatibility. The aim of this study is to construct an adjuvant delivery system (LNTPP/OVA) (Lentinan PLGA-stabilized Pickering emulsion loading OVA antigen) which can enhance the immune activity of LNT and can together deliver model protein antigen ovalbumin (OVA) into the organism. The characterization of the LNTPP/OVA was demonstrated that the size of LNTPP/OVA was around 1050.68 nm and was stable to store at least 28 days. Pickering emulsion was spherical shape like the raspberry with the high antigen load rate at around 82.53%. Moreover, the adjuvant effect of LNTPP/OVA formulation was detected. Compared with LNT/OVA formulation, our experimental results showed that LNTPP/OVA could promote the uptake of the OVA-antigen by macrophages in vitro. In vivo experiments, LNTPP/OVA facilitated the activation of dendritic cells (DCs) and induced strong humoral and cellular immune responses carrying a Th1 and Th2 immune responses. Therefore, LNTPP/OVA formulation have the latent capacity as a vaccine transmission system.

BacMam virus-based surface display for HCV E2 glycoprotein induces strong cross-neutralizing antibodies and cellular immune responses in vaccinated mice

BackgroundDespite recent advancements, limitations in the treatment and control of hepatitis C virus (HCV) infection reprioritized the studies for invention of an efficient HCV vaccine to elicit strong neutralizing antibodies (NAbs) and cellular responses.MethodsHerein, we report molecular construction of a BacMam virus-based surface display for a subtype-1a HCV gpE2 (Bac-CMV-E2-gp64; Bac) that both expressed and displayed gpE2 in mammalian cells and bacouloviral envelope, respectively.ResultsAssessments by western blotting, Immunofluorescence and Immunogold-electron microscopy indicated the proper expression and incorporation in insect cell and baculovirus envelope, respectively. Mice immunized in three different prime-boost immunization groups of: Bac/Bac, Bac/Pro (bacoulovirus-derived gpE2) and Bac/DNA (plasmid DNA (pCDNA)-encoding gpE2) developed high levels of IgG and IFN-γ (highest for Bac/Bac group) indicating the induction of both humeral and cellular immune responses. Calculation of the IgG2a/IgG1 and IFN-γ/IL-4 ratios indicated a Th1 polarization of immune responses in the Bac/Bac and Bac/DNA groups but a balanced Th1-Th2 phenotype in the Bac/Pro group. Sera of the mice in the Bac/Bac group provided the highest percentage of cross-NAbs against a subtype-2a HCVcc (JFH1) compared to Bac/Pro and Bac/DNA groups (62% versus 41% and 6%).ConclusionsResults indicated that BacMam virus-based surface display for gpE2 might act as both subunit and DNA vaccine and offers a promising strategy for development of HCV vaccine for concurrent induction of strong humoral and cellular immune responses.

A Combined Adjuvant TF-Al Consisting of TFPR1 and Aluminum Hydroxide Augments Strong Humoral and Cellular Immune Responses in Both C57BL/6 and BALB/c Mice.

TFPR1 is a novel adjuvant for protein and peptide antigens, which has been demonstrated in BALB/c mice in our previous studies; however, its adjuvanticity in mice with different genetic backgrounds remains unknown, and its adjuvanticity needs to be improved to fit the requirements for various vaccines. In this study, we first compared the adjuvanticity of TFPR1 in two commonly used inbred mouse strains, BALB/c and C57BL/6 mice, in vitro and in vivo, and demonstrated that TFPR1 activated TLR2 to exert its immune activity in vivo. Next, to prove the feasibility of TFPR1 acting as a major component of combined adjuvants, we prepared a combined adjuvant, TF–Al, by formulating TFPR1 and alum at a certain ratio and compared its adjuvanticity with that of TFPR1 and alum alone using OVA and recombinant HBsAg as model antigens in both BALB/c and C57BL/6 mice. Results showed that TFPR1 acts as an effective vaccine adjuvant in both BALB/c mice and C57BL/6 mice, and further demonstrated the role of TLR2 in the adjuvanticity of TFPR1 in vivo. In addition, we obtained a novel combined adjuvant, TF–Al, based on TFPR1, which can augment antibody and cellular immune responses in mice with different genetic backgrounds, suggesting its promise for vaccine development in the future.

ISCOM-like Nanoparticles Formulated with Quillaja brasiliensis Saponins Are Promising Adjuvants for Seasonal Influenza Vaccines.

Vaccination is the most effective public health intervention to prevent influenza infections, which are responsible for an important burden of respiratory illnesses and deaths each year. Currently, licensed influenza vaccines are mostly split inactivated, although in order to achieve higher efficacy rates, some influenza vaccines contain adjuvants. Although split-inactivated vaccines induce mostly humoral responses, tailoring mucosal and cellular immune responses is crucial for preventing influenza infections. Quillaja brasiliensis saponin-based adjuvants, including ISCOM-like nanoparticles formulated with the QB-90 saponin fraction (IQB90), have been studied in preclinical models for more than a decade and have been demonstrated to induce strong humoral and cellular immune responses towards several viral antigens. Herein, we demonstrate that a split-inactivated IQB90 adjuvanted influenza vaccine triggered a protective immune response, stronger than that induced by a commercial unadjuvanted vaccine, when applied either by the subcutaneous or the intranasal route. Moreover, we reveal that this novel adjuvant confers up to a ten-fold dose-sparing effect, which could be crucial for pandemic preparedness. Last but not least, we assessed the role of caspase-1/11 in the generation of the immune response triggered by the IQB90 adjuvanted influenza vaccine in a mouse model and found that the cellular-mediated immune response triggered by the IQB90-Flu relies, at least in part, on a mechanism involving the casp-1/11 pathway but not the humoral response elicited by this formulation.

An open, non-randomised, phase 1/2 trial on the safety, tolerability, and immunogenicity of single-dose vaccine "Sputnik Light" for prevention of coronavirus infection in healthy adults.

SummaryBackgroundWhile the world is experiencing another wave of COVID-19 pandemic, global vaccination program is hampered by an evident shortage in the supply of licensed vaccines. In an effort to satisfy vaccine demands we developed a new single-dose vaccine based on recombinant adenovirus type 26 (rAd26) vector carrying the gene for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein – “Sputnik Light”.MethodsWe conducted an open label, prospective, non-randomised phase 1/2 trial aimed to assess safety, tolerability, and immunogenicity of “Sputnik Light” vaccine in a single center in Russia. Primary outcome measures were antigen-specific humoral immunity (Anti-RBD-SARS-CoV-2 antibodies measured by ELISA on days 1, 10, 28, and 42) and safety (number of participants with adverse events monitored throughout the study). Secondary outcome measures were antigen-specific cellular immunity (measured by antigen-dependent CD4+ and CD8+ T-cell proliferation, number of antigen-specific interferon-γ-producing cells as well as interferon-γ concentration upon antigen restimulation) and change in neutralizing antibodies (measured in SARS-CoV-2 neutralization assay).FindingsMost of the solicited adverse reactions were mild (66·4% from all vaccinees), few were moderate (5·5%). No serious adverse events were detected. Assessment of Anti-RBD-SARS-CoV-2 antibodies revealed a group with pre-existing immunity to SARS-CoV-2. Upon this finding we separated all safety and immunogenicity data based on pre-existing immunity to SARS-CoV-2. There were notable differences in the vaccine effects on immunogenicity by the groups. Vaccination of seropositive (N=14) volunteers rapidly boosted RBD-specific IgGs from reciprocal geometric mean titer (​GMT) 594·4 at a baseline up to 26899 comparing to 29·09 in seronegative group (N=96) by day 10. By day 42 seroconversion rate reached 100% (93/93) in seronegative group with GMT 1648. At the same time, in the seropositive group, seroconversion rate by day 42 was 92·9% (13/14) with GMT 19986. Analysis of neutralizing antibodies to SARS-CoV-2 showed 81·7% (76/93) and 92·9% (13/14) seroconversion rates by day 42 with median reciprocal GMT 15·18 and 579·7 in the seronegative and seropositive groups, respectively. Antigen-specific T cell proliferation, formation of IFNy-producing cells, and IFNy secretion were observed in 96·7% (26/27), 96% (24/25), and 96% (24/25) of the seronegative group respectively and in 100% (3/3), 100% (5/5), and 100% (5/5) of the seropositive vaccinees, respectively.InterpretationThe single-dose rAd26 vector-based COVID-19 vaccine “Sputnik Light” has a good safety profile and induces a strong humoral and cellular immune responses both in seronegative and seropositive participants.FundingRussian Direct Investment Fund.

Alum and metoclopramide synergistically enhance cellular and humoral immunity after immunization with heat-killed Salmonella typhimurium vaccine

Typically, the killed form of microorganisms in combination with alum does not produce strong cellular immune responses. A recent investigation has indicated the role of dopamine D2 receptor antagonists like metoclopramide in reducing the polarization of immune responses toward Th2 immunity. This study was performed to evaluate the effects of a combination of alum and metoclopramide on the induction of cellular and humoral immunity in response to a heat-killed preparation ofSalmonella typhimurium(HKST). Wistar rats were immunized with the HKST vaccine alone or in combination with alum, metoclopramide, or the alum-metoclopramide mixture twice with a two-week interval. Fourteen days after the last vaccination, immune responses against S. typhimurium and the protective potential of the vaccines were assessed. The combination of alum and metoclopramide as an adjuvant augmented the potential of the HKST vaccine to enhance lymphocyte proliferation, delayed-type hypersensitivity reaction, and antibody titer. These results were concurrent with the polarization of immune response towards the Th1 response and improving protective immunity against S. typhimurium. Overall, the combination of alum and metoclopramide as an adjuvant synergistically enhanced cellular and humoral immunity after immunization with the HKST vaccine.

RGS5 Determines Neutrophil Migration in the Acute Inflammatory Phase of Bleomycin-Induced Lung Injury

The regulator of G protein signaling (RGS) represents a widespread system of controllers of cellular responses. The activities of the R4 subfamily of RGSs have been elucidated in allergic pulmonary diseases. However, the R4 signaling in other inflammatory lung diseases, with a strong cellular immune response, remained unexplored. Thus, our study aimed to discern the functional relevance of the R4 family member, RGS5, as a potential modulating element in this context. Gene profiling of the R4 subfamily showed increased RGS5 expression in human fibrosing lung disease samples. In line with this, RGS5 was markedly increased in murine lungs following bleomycin injury. RGS knock-out mice (RGS-/-) had preserved lung function while control mice showed significant combined ventilatory disorders three days after bleomycin application as compared to untreated control mice. Loss of RGS5 was associated with a significantly reduced neutrophil influx and tissue myeloperoxidase expression. In the LPS lung injury model, RGS5-/- mice also failed to recruit neutrophils into the lung, which was accompanied by reduced tissue myeloperoxidase levels after 24 h. Our in-vitro assays showed impaired migration of RGS5-/- neutrophils towards chemokines despite preserved Ca2+ signaling. ERK dephosphorylation might play a role in reduced neutrophil migration in our model. As a conclusion, loss of RGS5 preserves lung function and attenuates hyperinflammation in the acute phase of bleomycin-induced pulmonary fibrosis and LPS-induced lung injury. Targeting RGS5 might alleviate the severity of exacerbations in interstitial lung diseases.

SARS-COV-2 recombinant Receptor-Binding-Domain (RBD) induces neutralizing antibodies against variant strains of SARS-CoV-2 and SARS-CoV-1

SARS-CoV-2 is the etiological agent of COVID19. There are currently several licensed vaccines approved for human use and most of them target the spike protein in the virion envelope to induce protective immunity. Recently, variants that spread more quickly have emerged. There is evidence that some of these variants are less sensitive to neutralization in vitro, but it is not clear whether they can evade vaccine induced protection. In this study, we tested SARS-CoV-2 spike RBD as a vaccine antigen and explored the effect of formulation with Alum/MPLA or AddaS03 adjuvants. Our results show that RBD induces high titers of neutralizing antibodies and activates strong cellular immune responses. There is also significant cross-neutralization of variants B.1.1.7 and B.1.351 and to a lesser extent, SARS-CoV-1. These results indicate that recombinant RBD can be a viable candidate as a stand-alone vaccine or as a booster shot to diversify our strategy for COVID19 protection.

Yeast Shells Encapsulating Adjuvant AS04 as an Antigen Delivery System for a Novel Vaccine against Toxoplasma Gondii.

Toxoplasma gondii (T. gondii) infection causes severe zoonotic toxoplasmosis, which threatens the safety of almost one-third of the human population globally. However, there is no effective protective vaccine against human toxoplasmosis. This necessitates anti-T. gondii vaccine development, which is a main priority of public health. In this study, we optimized the adjuvant system 04 (AS04), a vaccine adjuvant constituted by 3-O-desacyl-4'-monophosphoryl lipid A (a TLR4 agonist) and aluminum salts, by packing it within natural extracts of β-glucan particles (GPs) from Saccharomyces cerevisiae to form a GP-AS04 hybrid adjuvant system. Through a simple mixing procedure, we loaded GP-AS04 particles with the total extract (TE) of T. gondii lysate, forming a novel anti-T. gondii vaccine GP-AS04-TE. Results indicated that the hybrid adjuvant can efficiently and stably load antigens, mediate antigen delivery, facilitate the dendritic uptake of antigens, boost dendritic cell maturation and stimulation, and increase the secretion of pro-inflammatory cytokines. In the mouse inoculation model, GP-AS04-TE significantly stimulated the function of dendritic cells, induced a very strong TE-specific humoral and cellular immune response, and finally showed a strong and effective protection against toxoplasma chronic and acute infections. This work proves the potential of GP-AS04 for exploitation as a vaccine against a range of pathogens.

Immunogenicity and reactogenicity of heterologous ChAdOx1 nCoV-19/mRNA vaccination

Heterologous priming with the ChAdOx1 nCoV-19 vector vaccine followed by boosting with a messenger RNA vaccine (BNT162b2 or mRNA-1273) is currently recommended in Germany, although data on immunogenicity and reactogenicity are not available. In this observational study we show that, in healthy adult individuals (n = 96), the heterologous vaccine regimen induced spike-specific IgG, neutralizing antibodies and spike-specific CD4 T cells, the levels of which which were significantly higher than after homologous vector vaccine boost (n = 55) and higher or comparable in magnitude to homologous mRNA vaccine regimens (n = 62). Moreover, spike-specific CD8 T cell levels after heterologous vaccination were significantly higher than after both homologous regimens. Spike-specific T cells were predominantly polyfunctional with largely overlapping cytokine-producing phenotypes in all three regimens. Recipients of both the homologous vector regimen and the heterologous vector/mRNA combination reported greater reactogenicity following the priming vector vaccination, whereas heterologous boosting was well tolerated and comparable to homologous mRNA boosting. Taken together, heterologous vector/mRNA boosting induces strong humoral and cellular immune responses with acceptable reactogenicity profiles.

Implementation of Adenovirus-Mediated Pulmonary Expression of Human ACE2 in HLA Transgenic Mice Enables Establishment of a COVID-19 Murine Model for Assessment of Immune Responses to SARS-CoV-2 Infection.

HLA transgenic mice are instrumental for evaluation of human-specific immune responses to viral infection. Mice do not develop COVID-19 upon infection with SARS-CoV-2 due to the strict tropism of the virus to the human ACE2 receptor. The aim of the current study was the implementation of an adenovirus-mediated infection protocol for human ACE2 expression in HLA transgenic mice. Transient pulmonary expression of the human ACE2 receptor in these mice results in their sensitisation to SARS-CoV-2 infection, consequently providing a valuable animal model for COVID-19. Infection results in a transient loss in body weight starting 3 days post-infection, reaching 20–30% loss of weight at day 7 and full recovery at days 11–13 post-infection. The evolution of the disease revealed high reproducibility and very low variability among individual mice. The method was implemented in two different strains of HLA immunized mice. Infected animals developed strong protective humoral and cellular immune responses specific to the viral spike-protein, strictly depending on the adenovirus-mediated human ACE2 expression. Convalescent animals were protected against a subsequent re-infection with SARS-CoV-2, demonstrating that the model may be applied for assessment of efficacy of anti-viral immune responses.

An Open, Non-Randomised, 1/2 Phase Study on the Safety, Tolerability, and Immunogenicity of Single Dose 'Sputnik Light' Vaccine for Prevention of Coronavirus Infection in Healthy Adults

Background: While the world is experiencing another wave of COVID-19 pandemic, global vaccination program is hampered by obvious shortage in supply of licensed vaccines. Development of new vaccines that are easy to manufacture and administer is highly desirable to overcome hurdles in vaccine scaling up and distribution, especially in developing countries. In the effort to satisfy vaccine demands we developed a new single dose vaccine based on recombinant adenovirus type 26 (rAd26) vector carrying the gene for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein – “Sputnik Light”.Methods: We conducted an open label, two-stage, prospective, non-randomized phase I-II trial aimed to assess safety, tolerability and immunogenicity of “Sputnik Light” vaccine in a single center in Russia. Primary outcome measures were antigen-specific humoral immunity (Anti-RBD-SARS-Cov2 antibodies measured by ELISA on days 1, 10, 28, and 42) and safety (number of participants with adverse events monitored throughout the study). Secondary outcome measures were antigen-specific cellular immunity (measured by antigen-dependent CD4+ and CD8+ T-cell proliferation, number of antigen-specific interferon-γ-producing cells as well as interferon-γ concentration upon antigen restimulation) and change in neutralizing antibodies (measured in SARS-CoV-2 neutralization assay). The trial is registered at ClinicalTrials.gov (NCT04713488).Findings: Between January 14 and 27, 2021, 150 participants were screened, of whom 110 were enrolled. Most of solicited adverse reactions were mild (66.4% from all vaccinees), few were moderate (5.5%). No serious adverse events were detected. Assessment of Anti-RBD-SARS-Cov2 antibodies revealed group with pre-existing immunity to SARS-CoV-2. Upon this finding we separated all safety and immunogenicity data according to pre-existing immunity to SARS-Cov2. There were notable differences in the vaccine effects on immunogenicity by the groups. Vaccination of seropositive (N=14) volunteers rapidly boosted RBD-specific IgGs from reciprocal geometric mean titer (​GMT) 594.4 at a baseline up to 26899 comparing to 29.09 in seronegative group (N=96) by day 10. By day 42 seroconversion rate reached 100% (93/93) in seronegative group with GMT 1648. At the same time in seropositive group seroconversion rate by day 42 was 92.9% (13/14) with GMT 19986. Analysis of neutralizing antibodies to SARS-CoV-2 showed 81.7% (76/93) and 92.9% (13/14) seroconversion rates by day 42 with median reciprocal GMT 15.18 and 579.7 in the seronegative and seropositive groups, respectively. Each test to assess cell-mediated immune reactions was carried out on a separate group of 30 volunteers on day 10 upon vaccination. Antigen-specific T cell proliferation, formation of IFNy-producing cells, as well as IFNy secretion were detected in 96.7% (26/27), 96% (24/25) and 96% (24/25) seronegative and in 100% (3/3) 100% (5/5) and 100% (5/5) seropositive vaccinees, correspondingly.Interpretation: The single dose rAd26 vector-based COVID-19 vaccine “Sputnik Light” has a good safety profile and induced strong humoral and cellular immune responses both in seronegative and seropositive participants. Further investigation is needed to assess the effectiveness of this vaccine against COVID-19 used for primary and secondary vaccination.Clinical Trial Registration Details: The trial is registered at ClinicalTrials.gov (NCT04713488).Funding Information: Russian Direct Investment Fund.Declaration of Interests: OVZ, TAO, IVD, OP, DVS, DMG, ASD, AIT, DNS, IBE, EAT, AGB, ASE, FMI, NAN, NLL, ASS, SVB, BSN, DYL, ALG report patents for an immunobiological expression vector, pharmaceutical agent, and its method of use to prevent COVID-19. All other authors declare no competing interests.Ethics Approval Statement: The trial was approved by the local ethic committee and was conducted with the approval of the Ministry of Health of Russian Federation in compliance with International Conference on Harmonization and National Good Clinical Practice guidelines and Declaration of Helsinki.

Western equine encephalitis virus virus-like particles from an insect cell-baculovirus system elicit the strong immune responses in mice.

Western equine encephalitis virus (WEEV) causes lethal encephalitis in humans and equines, and it poses a serious public health threat in many countries. Therefore, the development of an efficient vaccine remains an important challenge for the prevention of WEEV infection. This study presents the first description of WEEV virus-like particles (VLPs) generated from insect cells using recombinant baculoviruses. WEEV VLPs with 206 adjuvant could trigger a strong cellular immune response; increase the levels of IL-2, IL-4 and IFN-γ; and induce a high level of neutralizing antibodies against WEEV in mice. These data showed that the insect cell-baculovirus system is suitable for the production of WEEV VLPs and that these VLPs could elicit the strong immunogenicity in mice. These results suggest a new, nonreplicating, and effective vaccine candidate against WEEV infection.

Liver-Directed AAV8 Booster Vaccine Expressing Plasmodium falciparum Antigen Following Adenovirus Vaccine Priming Elicits Sterile Protection in a Murine Model.

Hepatocyte infection by malaria sporozoites is a bottleneck in the life-cycle of Plasmodium spp. including P. falciparum, which causes the most lethal form of malaria. Therefore, developing an effective vaccine capable of inducing the strong humoral and cellular immune responses necessary to block the pre-erythrocytic stage has potential to overcome the spatiotemporal hindrances pertaining to parasite biology and hepatic microanatomy. We recently showed that when combined with a human adenovirus type 5 (AdHu5)-priming vaccine, adeno-associated virus serotype 1 (AAV1) is a potent booster malaria vaccine vector capable of inducing strong and long-lasting protective immune responses in a rodent malaria model. Here, we evaluated the protective efficacy of a hepatotropic virus, adeno-associated virus serotype 8 (AAV8), as a booster vector because it can deliver a transgene potently and rapidly to the liver, the organ malaria sporozoites initially infect and multiply in following sporozoite injection by the bite of an infected mosquito. We first generated an AAV8-vectored vaccine expressing P. falciparum circumsporozoite protein (PfCSP). Intravenous (i.v.) administration of AAV8-PfCSP to mice initially primed with AdHu5-PfCSP resulted in a hepatocyte transduction rate ~2.5 times above that seen with intramuscular (i.m.) administration. This immunization regimen provided a better protection rate (100% sterile protection) than that of the i.m. AdHu5-prime/i.m. AAV8-boost regimen (60%, p < 0.05), i.m. AdHu5-prime/i.v. AAV1-boost (78%), or i.m. AdHu5-prime/i.m. AAV1-boost (80%) against challenge with transgenic PfCSP-expressing P. berghei sporozoites. Compared with the i.m. AdHu5-prime/i.v. AAV1-boost regimen, three other regimens induced higher levels of PfCSP-specific humoral immune responses. Importantly, a single i.v. dose of AAV8-PfCSP recruited CD8+ T cells, especially resident memory CD8+ T cells, in the liver. These data suggest that boost with i.v. AAV8-PfCSP can improve humoral and cellular immune responses in BALB/c mice. Therefore, this regimen holds great promise as a next-generation platform for the development of an effective malaria vaccine.

GRA8 DNA vaccine formulations protect against chronic toxoplasmosis

Toxoplasma gondii has a very wide host range and infects all warm-blooded animals including humans. The disease causes great economic losses both in animals and humans. Vaccination is the most effective approach to fight against toxoplasmosis however an effective vaccine has not been developed yet. In the present study, GRA8 protein of T. gondii that showed high immunogenicity in our previous microarray screening study was used to develop a DNA vaccine using pcDNA 3.3 vector for the first time. In order to increase the potency of the DNA vaccine, 10 times lower amount of GRA8 DNA vaccine was combined with molecular adjuvant CpG and formulated into a commercial liposome (pcDNA3.3-GRA8+CpG+Escort). Mice were vaccinated intramuscularly two times at three-week intervals and challenged orally with the T. gondii PRU strain tissue cysts. The humoral immune response was determined by Western Blot and ELISA. The cellular immune response was analyzed by flow cytometry, cytokine ELISA and MTT assay. Among the vaccine groups, pcDNA3.3-GRA8 and pcDNA3.3-GRA8+CpG+Escort induced strong IgG response compared to controls (P < 0.001). The IgG1 and IgG2a responses showed a balanced Th1–Th2 polarization. The ratio of CD4+ and CD8+ T lymphocytes secreting IFN-γ increased, and significantly higher extracellular IFN-γ secretion was achieved compared to the controls (P < 0.01). The amount of tissue cysts in the group of mice vaccinated with pcDNA3.3-GRA8 decreased significantly compared to control groups (P < 0.0001). In the group vaccinated with pcDNA3.3-GRA8+CpG+Escort, the amount of tissue cysts also decreased significantly compared to PBS (P = 0.0086) and Empty plasmid+CpG+Escort (P = 0.0007) groups. This study showed for the first time that pcDNA 3.3. vector encoding GRA8 with or without CpG and Liposome can induce strong cellular and humoral immune responses and confer strong protection against mouse model of chronic toxoplasmosis.

Aqueous extracts from cultivated Cistanche deserticola Y.C. Ma as polysaccharide adjuvant promote immune responses via facilitating dendritic cell activation

Ethnopharmacological relevanceHerbal polysaccharides have exhibited great immune-enhancing potential. Adjuvants are a key tool for developing efficacious vaccines. In our previous study, a water-soluble polysaccharide extracted from wild Cistanche deserticola Y.C. Ma showed potent immunostimulatory activity. Aim of studyIn this study, the immune profiles and efficacy of aqueous extracts of cultivated Cistanche deserticola Y.C. Ma (AECCD) on ICR mice against ovalbumin (OVA) were investigated. In vitro experiments, the possible DC activation mechanism by AECCD was evaluated. Materials and methodsAECCD were extracted using hot water after which the crude polysaccharides were precipitated by ethanol. Mice were firstly immunized subcutaneously with OVA (10 μg per mouse) alone or OVA (10 μg per mouse) respectively containing different dose of AECCD (200, 400 and 800 μg per mouse) on Days 1 and 14 and the magnitude and kinetics of antibodies and cell-mediated responses were then assessed. ResultsAECCD elicited vigorous and long-term IgG responses with mixed Th1/Th2 responses and up-regulated levels of Th-associated cytokines (CD4+IL-4, CD4+IFN-γ and CD8+IFN-γ). Moreover, AECCD induced the strong cellular immune response characterized by increased splenocyte proliferation as well as the activated T cell response. Notably, AECCD significantly enhanced the maturation of dendritic cells (DCs) and inhibited Tregs. In vitro experiments, Preliminary tests indicated that AECCD induced DC activation by promoting phenotypic maturation, cytokine section and allostimulatory activity. Toll-like receptor 4 (TLR4) was an essential receptor for DCs to directly bind AECCD. The inhibitors of NF-κB decreased the expression levels of CD40, CD80, CD86 and MHC-II and the production of IFN-γ, TNF-α and IL-6 through DCs. ConclusionsFinally, these findings suggested that AECCD could elicit potent and durable antigen specific immune responses through DC activation, which was involved in the regulation of maturation markers and cytokine expression via TLR4-related NF-κB pathway. The study indicates that AECCD is a potential immunomodulator.

Development and characterization of a HCMV multiantigen therapeutic vaccine for GBM using the UNITE platform.

e14565 Background: Glioblastoma (GBM) is an aggressive form of brain cancer with a median survival of 15 months which has remained unchanged despite technological advances in the standard of care. GBM cells specifically express human cytomegalovirus (HCMV) proteins providing a unique opportunity for targeted therapy. Methods: We utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) that codes for the HCMV proteins- pp65, gB and IE-1. The UNITE platform involves lysosomal targeting technology, fusing lysosome-associated protein 1 (LAMP1) with target antigens resulting in increased antigen presentation by MHC-I and II. ELISpot, flow cytometry and ELISA techniques were used to evaluate the vaccine immunogenicity and a syngeneic, orthotopic GBM mouse model that expresses HCMV proteins was used for efficacy studies. The tumor microenvironment studies were done using flow cytometry and MSD assay. Results: ITI-1001 vaccination showed a robust antigen-specific CD4 and CD8 T cell response in addition to a strong humoral response. Using GBM mouse model, therapeutic treatment of ITI-1001 vaccine resulted in ̃56% survival with subsequent long-term immunity. Investigating the tumor microenvironment showed significant CD4 T cell infiltration as well as enhanced Th1 and CD8 T cell activation. Regulatory T cells were also upregulated upon ITI-1001 vaccination and would be an attractive target to further improve this therapy. In addition, tumor burden negatively correlated with number of activated CD4 T cells (CD4 IFNγ+) reiterating the importance of CD4 activation in ITI-1001 efficacy and potentially identifying treatment responders and non-responders. Further characterization of these two groups showed high infiltration of CD3+, CD4+ and CD8+ T cells in responders compared with non- responders along with higher CD8 T cell activation. Conclusions: Thus, we show that vaccination with HCMV antigens using the ITI-1001-UNITE platform generates strong cellular and humoral immune responses, triggering significant anti-tumor activity that leads to enhanced survival in mice with GBM.