In situ vaccination (ISV) triggers antitumor immune responses using the patient’s own cancer antigens, yet limited neoantigen release hampers its efficacy. Our novel combination therapy involves low-dose local cisplatin followed by ISV with a TLR7/8/9 agonist formulation (CR108), in which CR108 boosts and sustains the antitumor responses induced by the cisplatin-released neoantigens. In mouse models, the cisplatin+CR108 combination significantly outperformed cisplatin or CR108 alone in abrogating established 4T1 and B16 tumors. The synergistic antitumor effects of cisplatin and CR108 were accompanied by markedly increased tumor tertiary lymphatic structures (TLS) formation, higher levels of type I and III interferons and TNF-α in serum, augmented T and B lymphocyte infiltration, antigen-presenting cell activation, as well as reduced functionally of exhausted T cells. Single-cell sequencing analysis uncovered a potential pathway for TLS to serve as a reservoir for functional antitumor effector T cells. Furthermore, cisplatin+CR108 combo therapy, but neither cisplatin nor CR108 alone, effectively inhibited the growth of treated 4T-1 tumor in an effector T cell-dependent manner. Notably, the combo therapy also suppressed the growth of distant untreated 4T-1 tumors, demonstrating systemic antitumor effects. Moreover, combo-therapy led to full regression of 4T-1 tumors in a large percentage of mice, who became strongly resistant to secondary tumor challenge, a clear indication of antitumor immunological memory. The cisplatin+CR108 combo therapy holds promise in converting “cold” tumors into “hot” ones and eliciting robust antitumor immune responses in vivo.

Breakthrough infections by SARS-CoV-2 variants pose a global challenge to COVID-19 pandemic control, and the development of more effective vaccines of broad-spectrum protection is needed. In this study, we constructed pVAX1-based plasmids encoding receptor-binding domain (RBD) chimera of SARS-CoV-1 and SARS-CoV-2 variants, including pAD1002 (encoding RBD^SARS/BA1), pAD1003 (encoding RBD^SARS/Beta) and pAD131 (encoding RBD^BA1/Beta). Plasmids pAD1002 and pAD131 were far more immunogenic than pAD1003 in terms of eliciting RBD-specific IgG when intramuscularly administered without electroporation. Furthermore, dissolvable microneedle array patches (MAP) greatly enhanced the immunogenicity of these DNA constructs in mice and rabbits. MAP laden with pAD1002 (MAP-1002) significantly outperformed inactivated SARS-CoV-2 virus vaccine in inducing RBD-specific IFN-g⁺ effector and memory T cells, and generated T lymphocytes of different homing patterns compared to that induced by electroporated DNA in mice. In consistence with the high titer neutralization results of MAP-1002 antisera against SARS-CoV-2 pseudoviruses, MAP-1002 protected human ACE2-transgenic mice from Omicron BA.1 challenge. Collectively, MAP-based DNA constructs encoding chimeric RBDs of SARS-CoV-1 and SARS-CoV-2 variants, as represented by MAP-1002, are potential COVID-19 vaccine candidates worthy further translational study.

Waves of breakthrough infections by SARS-CoV-2 Omicron subvariants currently pose a global challenge to the control of the COVID-19 pandemic. We previously reported a pVAX1-based DNA vaccine candidate, pAD1002, that encodes a receptor-binding domain (RBD) chimera of SARS-CoV-1 and Omicron BA.1. In mouse and rabbit models, pAD1002 plasmid induced cross-neutralizing Abs against heterologous sarbecoviruses, including SARS-CoV-1 and SARS-CoV-2 wildtype, Delta and Omicron variants. However, these antisera failed to block the recent emerging Omicron subvariants BF.7 and BQ.1. To solve this problem, we replaced the BA.1 RBD-encoding DNA sequence in pAD1002 with that of BA.4/5. The resulting construct, namely pAD1016, elicited SARS-CoV-1 and SARS-CoV-2 RBD-specific IFN-γ⁺ cellular responses in BALB/c and C57BL/6 mice. More importantly, pAD1016 vaccination in mice, rabbits and pigs generated serum Abs capable of neutralizing pseudoviruses representing multiple SARS-CoV-2 Omicron subvariants including BA.2, BA.4/5, BF.7, BQ.1 and XBB. As a booster vaccine for inactivated SARS-CoV-2 virus preimmunization in mice, pAD1016 broadened the serum Ab neutralization spectrum to cover the Omicron BA.4/5, BF7 and BQ.1 subvariants. These preliminary data highlight the potential benefit of pAD1016 in eliciting neutralizing Abs against broad-spectrum Omicron subvariants in individuals previously vaccinated with inactivated prototype SARS-CoV-2 virus and suggests that pAD1016 is worthy of further translational study as a COVID-19 vaccine candidate.

The SARS-CoV-2 pandemic remains an ongoing threat to global health with emerging variants, especially the Omicron variant and its sub-lineages. Although large-scale vaccination worldwide has delivered outstanding achievements for COVID-19 prevention, a declining effectiveness to a different extent in emerging SARS-CoV-2 variants was observed in the vaccinated population. Vaccines eliciting broader spectrum neutralizing antibodies and cellular immune responses are urgently needed and important. To achieve this goal, rational vaccine design, including antigen modeling, screening and combination, vaccine pipelines, and delivery, are keys to developing a next-generation COVID-19 vaccine. In this study, we designed several DNA constructs based on codon-optimized spike coding regions of several SARS-CoV-2 variants and analyzed their cross-reactive antibodies, including neutralizing antibodies, and cellular immune responses against several VOCs in C57BL/6 mice. The results revealed that different SARS-CoV-2 VOCs induced different cross-reactivity; pBeta, a DNA vaccine encoding the spike protein of the Beta variant, elicited broader cross-reactive neutralizing antibodies against other variants including the Omicron variants BA.1 and BA.4/5. This result demonstrates that the spike antigen from the Beta variant potentially serves as one of the antigens for multivalent vaccine design and development against variants of SARS-CoV-2.

Waves of breakthrough infections by SARS-CoV-2 Omicron subvariants pose a global challenge to pandemic control today. We have previously reported a pVAX1-based DNA vaccine candidate, pAD1002, which encodes a receptor-binding domain (RBD) chimera of SARS-CoV-1 and Omicron BA.1. In mouse and rabbit models, pAD1002 plasmid induced cross-neutralizing Abs against heterologous Sarbecoviruses, including SARS-CoV-1 and SARS-CoV-2 prototype, Delta and Omicron variants. However, these antisera failed to block the recent emerging Omicron subvariants BF.7 and BQ.1. To solve this problem, we replaced the BA.1-encoding DNA sequence in pAD1002 with that of BA.4/5. The resulting construct, namely pAD1016, elicited SARS-CoV-1 and SARS-CoV-2 RBD-specific IFN-γ ⁺ cellular responses in BALB/c and C57BL/6 mice. More importantly, pAD1016 vaccination in mice and rabbits generated serum Abs capable of neutralizing pseudoviruses representing multiple SARS-CoV-2 Omicron subvariants including BA.2, BA.4/5, BF.7, BQ.1 and XBB. As a booster vaccine for inactivated SARS-CoV-2 virus preimmunization in C57BL/6 mice, pAD1016 broadened the serum Ab neutralization spectrum to cover the Omicron BA.4/5, BF7 and BQ.1 subvariants. These data highlight the potential benefit of pAD1016 in eliciting neutralizing Abs against broad spectrum Omicron subvariants in individuals previously vaccinated with inactivated prototype SARS-CoV-2 virus and suggests that pAD1016 is worthy further translational study as a COVID-19 vaccine candidate.

Breakthrough infections by SARS-CoV-2 variants pose a global challenge to pandemic control, and the development of more effective vaccines of broadspectrum protection is needed. In this study, we constructed pVAX1-based plasmids encoding heterodimeric receptor-binding domain (RBD) chimera of SARS-CoV and SARS-CoV-2 Omicron BA.1 (RBD ^SARS/BA1 ), SARS-CoV and SARS-CoV-2 Beta (RBD ^SARS/Beta ), or Omicron BA.1 and Beta (RBD ^BA1/Beta ) in secreted form. When i.m. injected in mice, RBD ^SARS/BA1 and RBD ^SARS/Beta encoding plasmids (pAD1002 and pAD131, respectively) were by far more immunogenic than RBD ^BA1/Beta plasmid (pAD1003). Dissolvable microneedle array patches (MAP) laden with these DNA plasmids were fabricated. All 3 resulting MAP-based vaccine candidates, namely MAP-1002, MAP1003 and MAP-131, were comparable to i.m. inoculated plasmids with electroporation assistance in eliciting strong and durable IgG responses in BALB/c and C57BL/6 mice as well as rabbits, while MAP-1002 was comparatively the most immunogenic. More importantly, MAP-1002 significantly outperformed inactivated SARS-CoV-2 virus vaccine in inducing RBD-specific IFN-γ ⁺ T cells. Moreover, MAP-1002 antisera effectively neutralized pseudoviruses displaying spike proteins of SARS-CoV, prototype SARS-CoV-2 or Beta, Delta, Omicron BA1, BA2 and BA4/5 variants. Collectively, MAP-based DNA constructs encoding chimeric RBDs of SARS-CoV and SARS-CoV-2 variants, as represented by MAP-1002, are potential COVID-19 vaccine candidates worthy further translational study.