OVA Peptide 323-339

CD4+ T-cell epitope-based heterologous prime-boost vaccination potentiates anti-tumor immunity and PD-1/PD-L1 immunotherapy

Background: Antitumor therapeutic vaccines are generally based on antigenic epitopes presented by major histocompatibility complex (MHC-I) molecules to induce tumor-specific CD8+ T cells. Paradoxically, continuous T cell receptor (TCR) stimulation from tumor-derived CD8+ T-cell epitopes can drive the functional exhaustion of tumor-specific CD8+ T cells. Tumor-specific type-I helper CD4+ T (TH1) cells play an important role in the population maintenance and cytotoxic function of exhausted tumor-specific CD8+ T cells in the tumor microenvironment. Nonetheless, whether the vaccination strategy targeting MHC-II-restricted CD4+ T-cell epitopes to induce tumor-specific TH1 responses can confer effective antitumor immunity to restrain tumor growth is not well studied. Here, we developed a heterologous prime-boost vaccination strategy to effectively induce tumor-specific TH1 cells and evaluated its antitumor efficacy and its capacity to potentiate PD-1/PD-L1 immunotherapy. Methods: Listeria monocytogenes vector and influenza A virus (PR8 strain) vector stably expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein-specific I-Ab-restricted CD4+ T cell epitope (GP61-80) or ovalbumin-specific CD4+ T cell epitope (OVA323-339) were constructed and evaluated their efficacy against mouse models of melanoma and colorectal adenocarcinoma expressing lymphocytic choriomeningitis virus glycoprotein and ovalbumin. The impact of CD4+ T cell epitope-based heterologous prime-boost vaccination was detected by flow-cytometer, single-cell RNA sequencing and single-cell TCR sequencing. Results: CD4+ T cell epitope-based heterologous prime-boost vaccination efficiently suppressed both mouse melanoma and colorectal adenocarcinoma. This vaccination primarily induced tumor-specific TH1 response, which in turn enhanced the expansion, effector function and clonal breadth of tumor-specific CD8+ T cells. Furthermore, this vaccination strategy synergized PD-L1 blockade mediated tumor suppression. Notably, prime-boost vaccination extended the duration of PD-L1 blockade induced antitumor effects by preventing the re-exhaustion of tumor-specific CD8+ T cells. Conclusion: CD4+ T cell epitope-based heterologous prime-boost vaccination elicited potent both tumor-specific TH1 and CTL response, leading to the efficient tumor control. This strategy can also potentiate PD-1/PD-L1 immune checkpoint blockade (ICB) against cancer.

Comparison between ovalbumin and ovalbumin peptide 323-339 responses in allergic mice: humoral and cellular aspects

Ovalbumin (OVA) is widely used in allergy research. OVA peptide 323-339 has been reported to be responsible for 25-35% of isolated BALB/c mouse T-cell response to intact OVA. An investigation of whether OVA and OVA 323-339 molecules can induce equivalent in vivo and in vitro immune responses was conducted. Eight-week-old BALB/c mice were randomly divided into three groups: OVA, OVA 323-339 and saline. On days 0, 7, 14, mice were intraperitoneally injected with 25 microg OVA or OVA 323-339 absorbed on 300 microg Alum, or saline; on days 21-23, all groups were challenged intranasally with either 20 microl of 1% OVA, 1% OVA 323-339 or saline. On day 28, after killing, splenocytes were isolated and cultured under the stimulus of each allergen or medium. Evaluated by hematoxylin/eosin and major basic protein immunohistochemical stainings, OVA and OVA 323-339 induced similar lung inflammation. Interestingly, significant serum total IgE and OVA-specific IgE were observed in OVA mice when compared to saline control. OVA 323-339 mice showed higher serum OVA-specific IgE, OVA 323-339-specific IgE, IL-4 and lower IFN-gamma similar to OVA mice. The proliferative response to OVA was found in cultured splenocytes of both OVA and OVA 323-339 mice, while the similar proliferative response to OVA 323-339 was only observed in the splenocytes of OVA 323-339-sensitized and challenged mice. Although OVA 323-339 induced a Th2-like response in the mouse model as did OVA, OVA 323-339 has clearly limited immunogenic potency to activate OVA-sensitized and challenged mice splenocytes, unlike OVA.

Ovalbumin(323-339) peptide binds to the major histocompatibility complex class II I-A(d) protein using two functionally distinct registers

Proteins of the class II major histocompatibility complex (MHC) bind antigenic peptides that are subsequently presented to T cells. Previous studies have shown that most of the residues required for binding of the chicken ovalbumin (Ova) 323-339 peptide to the I-A(d) MHC class II protein are contained within the shorter 325-336 peptide. This observation is somewhat inconsistent with the X-ray structure of the Ova peptide covalently attached to I-A(d) ( structure) in which residues 323 and 324 form binding interactions with the protein. A second register for the Ova(325-336) peptide is proposed where residues 326 and 327 occupy positions similar to residues 323 and 324 in the structure. Two Ova peptides that minimally encompass the and alternate registers, Ova(323-335) and Ova(325-336), respectively, were found to dissociate from I-A(d) with distinct kinetics. The dissociation rates for both peptides were enhanced when the His81 residue of the MHC beta-chain was replaced with an asparagine. In the structure the betaH81 residue forms a hydrogen bond to the backbone carbonyl of I323. If the Ova(325-336) peptide were also bound in the register, there would be no comparable hydrogen-bond acceptor for the betaH81 side chain that could explain this peptide's sensitivity to the betaH81 replacement. The Ova(323-335) peptide that binds in the register does not stimulate a T-cell hybridoma that is stimulated by Ova(325-336) bound in the alternate register. These results demonstrate that a single peptide can bind to an MHC peptide in alternate registers producing distinct T-cell responses.

The Serpin-like Loop Insertion of Ovalbumin Increases the Stability and Decreases the OVA 323-339 Epitope Processing Efficiency

Chicken ovalbumin (cOVA) has been studied for decades primarily due to the robust genetic and molecular resources that are available for experimental investigations. cOVA is a member of the serpin superfamily of proteins that function as protease inhibitors, although cOVA does not exhibit this activity. As a serpin, cOVA possesses a protease-sensitive reactive center loop that lies adjacent to the OVA 323-339 CD4+ T-cell epitope. We took advantage of the previously described single-substitution variant, OVA R339T, which can undergo the dramatic structural transition observed in serpins, to study how changes in loop size and protein stability influence the processing and presentation of the OVA 323-339 epitope. We observed that the OVA R339T loop insertion increases the stability and protease resistance, resulting in the reduced presentation of the OVA 323-339 epitope in vitro. These findings have implications for the design of more effective vaccines for the treatment of infectious diseases and cancer as well as the development of more robust CD4+ T-cell epitope prediction tools.

Comparison of the allergenicity of ovalbumin and ovalbumin peptide 323-339. Differential expansion of V beta-expressing T cell populations

We analyzed the effects of sensitization of BALB/c mice to the OVA peptide amino acids 323-339, on the development of an IgE response, immediate cutaneous hypersensitivity and airways responsiveness (AR). Daily aerosolization of OVA 323-339 for 20 min over a period of 10 days was as effective in the stimulation of a serum anti-OVA IgE antibody response as sensitization to native OVA by the same route. After sensitization to native OVA, the majority of the IgE anti-OVA response was directed against peptide 323-339. The antibody responses were paralleled by skin test responses in sensitized mice: 73% of OVA-sensitized mice developed immediate type reactions when tested with native OVA and 82% of the mice had positive immediate skin test responses to intradermal injection of peptide 323-339. After sensitization to the peptide, 69% of the mice had positive responses to native OVA and 77% responded to peptide 323-339. Aerosolization of OVA as well as OVA 323-339 led to a comparable increase in airway responsiveness as measured by electrical field stimulation of tracheal smooth muscle preparations. To characterize T cell subpopulations that were stimulated after allergen sensitization, the distribution of specific V beta-expressing T cells was analyzed in local draining lymph nodes of the airways and the lungs. These lymph nodes were found to be enlarged after both OVA and OVA peptide sensitization. Sensitization to native OVA resulted in an increased percentage of V beta 8.1 and V beta 8.2 T cells whereas selective stimulation of V beta 8.1 T cells was found after peptide sensitization. These data indicate that OVA peptide 323-339 represents a T and B cell epitope of OVA, which is important in the generation and development of immediate hypersensitivity responses in BALB/c mice.