Different formulations of inactivated SARS-CoV-2 vaccine candidates in human compatible adjuvants: Potency studies in mice showed different platforms of immune responses

Several inactivated SARS-CoV-2 vaccines were approved for human use but are not highly potent. Here, different formulations of inactivated SARS-CoV-2 virus in Alum, Montanide 51VG and Montanide ISA720VG adjuvants were developed and then immune responses were assessed. SARS-CoV-2 virus was inactivated with formalin and formulated in the adjuvants. BALB/c mice were immunized subcutaneously with 4µg of experimental vaccines on days 0 and 14 and two weeks after the final immunization, IL-4 and IFN-γ cytokines, CTL activity and specific IgG titer and IgG1, IgG2a, IgG2a/IgG1 ratio and also anti-RBD IgG response were assessed. Immunization with SARS-CoV-2-Montanide ISA51VG showed a significant increase in IFN-γ cytokine versus SARS-CoV-2-Alum, SARS-CoV-2-Montanide ISA720VG and control groups (P<0.0033). Cytokine IL-4 response in SARS-CoV-2-Alum group showed a significant increase versus SARS-CoV-2-Montanide ISA51VG, SARS-CoV-2-Montanide ISA720VG and control groups (P<0.0206). In addition, SARS-CoV-2-Montanide ISA51VG vaccine induced the highest IFN-γ/IL-4 cytokine ratio versus other groups (P<0.0004). CTL activity in SARS-CoV-2- Montanide ISA51 VG and SARS-CoV-2-Montanide ISA720 VG groups showed a significant increase versus SARS-CoV-2-Alum and control groups (P<0.0075). Specific IgG titer in SARS-CoV-2- Montanide ISA51 VG and SARS-CoV-2-Montanide ISA720VG showed significant increase versus SARS-CoV-2-Alum and control groups (P<0.0143). Results of specific IgG1and IgG2a level in SARS-COV-2-Alum, SARS-COV-2-Montanide ISA51VG and SARS-COV-2-Montanide ISA720VG vaccine showed a significant increase versus the control group (P<0.0001) but SARS-COV-2-Montanide ISA51VG and SARS-COV-2-Montanide ISA 720VG groups showed highest IgG2a/IgG1 ratio and a significant increase versus SARS-COV-2-Alum group (P<0.0379). Results of anti-RBD IgG response showed that inactivated SARS-COV-2+Alum and SARS-COV-2-Montanide ISA 720VG vaccine groups demonstrated a significant increase versus SARS-COV-2-Montanide ISA51VG group. It seems that the type of vaccine formulation is a critical parameter that effect on immunologic patterns and vaccine potency. Here, results showed that human compatible oil-based adjuvants were more potent than Alum adjuvant in the induction of cellular and humoral immune responses versus SARS-CoV-2 virus.

Flow Cytometric Analysis of the Cytotoxic T-Cell Recall Response to Theileria parva in Cattle Following Vaccination by the Infection and Treatment Method

The apicomplexan hemoparasite, Theileria parva, causes East Coast fever (ECF), a frequently fatal disease of African cattle. Vaccine development has been impeded by incomplete understanding of protective immunity following natural exposure or the infection and treatment method (ITM) of immunization. This is attributable to a paucity of methods to characterize the memory T-cell repertoire following infection. To overcome this impediment, assays developed to study the immune response to other intracellular pathogens were adapted for use in studies with T. parva to enable definition of the phenotype and function of effector T cells in T. parva-immune cattle, facilitating vaccine development. As reported herein, stimulation of peripheral blood mononuclear cells (PBMC) from ITM-immunized steers with irradiated, autologous, T. parva-infected cell lines elicited a proliferative recall response comprised of CD45R0+/CCR7− CD4+ and CD8+ T cells. Subsequent co-incubation of stimulated cultures with infected cells demonstrated the presence of cytotoxic T cells (CTLs) with the ability to kill infected cells. Comparison of CTL activity in cultures depleted of CD4+ or CD8+ T cells demonstrated CTL activity was primarily attributed to CD8+ T cells. Importantly, stimulation of PBMC from vaccinated steers always elicited proliferation of CD4+ and CD8+ T cells. This was the first important observation obtained from the use of the assay described herein.

The CD8+ T Cell Noncytotoxic Antiviral Responses

SUMMARY The CD8+ T cell noncytotoxic antiviral response (CNAR) was discovered during studies of asymptomatic HIV-infected subjects more than 30 years ago. In contrast to CD8+ T cell cytotoxic lymphocyte (CTL) activity, CNAR suppresses HIV replication without target cell killing. This activity has characteristics of innate immunity: it acts on all retroviruses and thus is neither epitope specific nor HLA restricted. The HIV-associated CNAR does not affect other virus families. It is mediated, at least in part, by a CD8+ T cell antiviral factor (CAF) that blocks HIV transcription. A variety of assays used to measure CNAR/CAF and the effects on other retrovirus infections are described. Notably, CD8+ T cell noncytotoxic antiviral responses have now been observed with other virus families but are mediated by different cytokines. Characterizing the protein structure of CAF has been challenging despite many biologic, immunologic, and molecular studies. It represents a low-abundance protein that may be identified by future next-generation sequencing approaches. Since CNAR/CAF is a natural noncytotoxic activity, it could provide promising strategies for HIV/AIDS therapy, cure, and prevention.

TIGIT expression on CD8+ T cells correlates with higher cytotoxic capacity

Persistent exposure to antigen leads to T cell exhaustion and immunologic dysfunction. We examined the immune exhaustion markers TIGIT and PD-1 in HIV-infected and healthy individuals and the relationship with cytotoxic CD8 + T lymphocyte (CTL) activity. Frequencies of TIGIT but not PD-1 positively correlated with CTL activity in HIV-aviremic and healthy individuals; however, there was no correlation in HIV-viremic individuals. Transcriptome analyses revealed upregulation of genes associated with antiviral immunity in TIGIT + versus TIGIT -CD8 + T cells. Our data suggest that TIGIT +CD8 + T cells do not necessarily represent a state of immune exhaustion and maintain an intrinsic cytotoxicity in HIV-infected individuals.

Phase II open-label study of S-588410 as maintenance monotherapy after first-line platinum-containing chemotherapy in patients with advanced or metastatic urothelial carcinoma.

440 Background: S-588410 is a cancer peptide vaccine composed of 5 human leukocyte antigen (HLA)-A*24:02-restricted epitope peptides derived from 5 cancer-testis antigens: DEPDC1, MPHOSPH1, URLC10, CDCA1 and KOC1; all of which are highly expressed in urothelial carcinoma. This study aimed to evaluate the effect of S-588410 maintenance therapy on peptide-specific cytotoxic T-lymphocyte (CTL) induction in patients with advanced or metastatic urothelial carcinoma after first-line platinum-based chemotherapy. Methods: An open-label, multicenter phase II trial was performed across 62 sites in Japan, the United Kingdom, France and Bulgaria (EudraCT 2013-005274-22). Eligible patients had completed ≥4 cycles of first-line platinum-based chemotherapy without disease progression. HLA-A*24:02-positive patients received S-588410 (1 mg of each of 5 peptides mixed with Montanide ISA 51 VG) subcutaneously weekly for 12 weeks, then every 2 weeks for up to 2 years. HLA-A*24:02-negative patients were enrolled in an observation group and did not receive study drug. The primary endpoint for the S-588410 group was the CTL induction rate at 12 weeks, defined as the proportion of patients who showed increased CTL activity for ≥1 peptide. Secondary endpoints included CTL induction rate after 1 year, antitumor effect defined by immune-related response criteria, progression-free survival (PFS), overall survival (OS), and safety. Results: A total of 81 patients with platinum-sensitive advanced or metastatic urothelial carcinoma were enrolled (S-588410 group, n=45; observation group, n=36) between April 2014 and November 2017. Most patients were male and Asian with a mean age of 67 years. CTLs were induced in 42 (93.3%) patients who received S-588410 for 12 weeks (P<0.0001, one-sided binomial test where the CTL induction rate is ≤50% as the null hypothesis). The CTL induction rate steadily increased to 95.6% within 48 weeks. CTL activity was high for the DEPDC1, MPHOSPH1 and URLC10 peptides. The response rate (immune-related complete response [CR] or partial response [PR]) was 8.9% (4/45 patients) in the S-588410 group and 0% in the observation group. Tumor imaging showed gradual (PR, n=3) and durable (CR, n=1) tumor shrinkage after ≥36 weeks in the S-588410 group. Median PFS was 18.1 weeks in the S-588410 group and 12.5 weeks in the observation group. Median OS was 71 and 99 weeks, respectively. The most frequent treatment-emergent adverse event was injection site reaction (42/45 patients [93.3%]; Grades 1–3). Pyrexia, rash and pruritus were also observed in the S-588410 group, but not the observation group. Conclusions: S-588410 showed a potent immune response and acceptable safety profile in patients with advanced or metastatic urothelial carcinoma, potentially offering a clinical benefit as post-chemotherapy maintenance therapy. Clinical trial information: EudraCT 2013-005274-22.

Evaluation of HIV-1 Regulatory and Structural Proteins as Antigen Candidate in Mouse and Human

Background:: The diagnosis of HIV infection is important among different groups. Moreover, combination antiretroviral therapy is used to treat HIV-1, but it cannot eradicate the infection. Thus, development of therapeutic vaccines along with antiretroviral therapy is recommended. This study evaluates the values of four HIV proteins as an antigen candidate in therapeutic vaccine design as well as a possible diagnostic marker for HIV infection in human. Methods:: In this study, the HIV-1 Tat and Rev regulatory proteins and structural Gp120 and p24 proteins were generated in E. coli expression system. Their immunogenicity was evaluated in BALB/c mice using homologous and heterologous prime/boost strategies. Moreover, the detection of anti-HIV IgG antibodies against these recombinant proteins was assessed in untreated (Naïve/ HIV-infected), treated and drug-resistant patients compared to healthy (control) group as a possible diagnostic marker for HIV infection. Results:: In human, our results showed that among HIV-1 proteins, anti-Gp120 antibody was not detected in treated individuals compared to healthy (control) group. The levels of anti-Gp120 antibody were significantly different between treated group and Naïve as well as drug-resistant subjects. Moreover, the level of anti-p24 antibody was significantly lower in treated group than Naive group. In mice, the results of immunization indicated that the Rev antigen could significantly induce IgG2a, IgG2b and IFN-γ secretion aimed at Th1 response as well as Granzyme B generation as CTL activity in comparison with other antigens. Furthermore, the heterologous DNA prime/ protein boost regimen was more potent than the homologous regimen for stimulation of cellular immunity. Conclusion:: Briefly, the levels of both anti-Gp120 and anti-p24 antibodies can be considered for diagnosis of the HIVinfected individuals in different groups compared to healthy group. Moreover, among four recombinant proteins, Rev elicited Th1 cellular immunity and CTL activity in mice as an antigen candidate in therapeutic vaccine development.

Internally quenched fluorogenic probe provides selective and rapid detection of cathepsin L activity

Cathepsin L (CTL) is a cysteine protease that demonstrates upregulated activity and/or altered trafficking during disease states such as cancer. The overlapping substrate specificity of cathepsin family members makes selective detection of activity from a single cathepsin difficult, and CTL activity is particularly difficult to parse from its close homologue CTV and the ubiquitous CTB. Despite this, screening campaigns have explored the extended chemical space in the cathepsin binding sites and identified unique substrate structures that offer selectivity for one enzyme over others. In this vein, we present CTLAP, a fluorogenic probe that is rapidly activated by CTL and displays good selectivity over CTB and CTV, the closest competing analytes for CTL activity probes. CTLAP exhibits intrinsically low background fluorescence, which we attribute to possible self-quenching mechanisms. CTLAP demonstrates markedly higher turn-on ratios (24-fold) and moderately improved enzyme selectivity compared to Z-FR-AMC (10-fold turn-on ratio), a commercially available CTL-selective probe commonly used to detect CTL activity in mixed samples. Optimum selectivity for CTL is achieved within 10 min of incubation with the enzyme, suggesting that CTLAP is amenable for rapid detection of CTL, even in the presence of competing cathepsins.