Nanoparticle Vaccines Based on the Receptor Binding Domain (RBD) and Heptad Repeat (HR) of SARS-CoV-2 Elicit Robust Protective Immune Responses.

Various vaccine strategies have been proposed in response to the global COVID-19 pandemic, each with unique strategies for eliciting immune responses. Here, we developed nanoparticle vaccines by covalently conjugating the self-assembled 24-mer ferritin to the receptor binding domain (RBD) and/or heptad repeat (HR) subunits of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) spike (S) protein. Compared to monomer vaccines, nanoparticle vaccines elicited more robust neutralizing antibodies and cellular immune responses. RBD and RBD-HR nanoparticle vaccinated hACE2 transgenic mice vaccinated with RBD and/or RBD-HR nanoparticles exhibited reduced viral load in the lungs after SARS-CoV-2 challenge. RBD-HR nanoparticle vaccines also promoted neutralizing antibodies and cellular immune responses against other coronaviruses. The nanoparticle vaccination of rhesus macaques induced neutralizing antibodies, and T and B cell responses prior to boost immunization; these responses persisted for more than three months. RBD- and HR-based nanoparticles thus present a promising vaccination approach against SARS-CoV-2 and other coronaviruses.

Pam2CSK4-adjuvanted SARS-CoV-2 RBD nanoparticle vaccine induces robust humoral and cellular immune responses.

As the global COVID-19 pandemic continues and new SARS-CoV-2 variants of concern emerge, vaccines remain an important tool for preventing the pandemic. The inactivated or subunit vaccines themselves generally exhibit low immunogenicity, which needs adjuvants to improve the immune response. We previously developed a receptor binding domain (RBD)-targeted and self-assembled nanoparticle to elicit a potent immune response in both mice and rhesus macaques. Herein, we further improved the RBD production in the eukaryote system by in situ Crispr/Cas9-engineered CHO cells. By comparing the immune effects of various Toll-like receptor-targeted adjuvants to enhance nanoparticle vaccine immunization, we found that Pam2CSK4, a TLR2/6 agonist, could mostly increase the titers of antigen-specific neutralizing antibodies and durability in humoral immunity. Remarkably, together with Pam2CSK4, the RBD-based nanoparticle vaccine induced a significant Th1-biased immune response and enhanced the differentiation of both memory T cells and follicular helper T cells. We further found that Pam2CSK4 upregulated migration genes and many genes involved in the activation and proliferation of leukocytes. Our data indicate that Pam2CSK4 targeting TLR2, which has been shown to be effective in tuberculosis vaccines, is the optimal adjuvant for the SARS-CoV-2 nanoparticle vaccine, paving the way for an immediate clinical trial.

Engineering of α-PD-1 antibody-expressing long-lived plasma cells by CRISPR/Cas9-mediated targeted gene integration.

Long-lived plasma cells (LLPCs) are robust specialized antibody-secreting cells that mainly stay in the bone marrow and can persist a lifetime. As they can be generated by inducing the differentiation of B-lymphocytes, we investigated the possibility that human LLPCs might be engineered to express α-PD-1 monoclonal antibody to substitute recombinant α-PD-1 antitumor immunotherapy. To this end, we inserted an α-PD-1 cassette into the GAPDH locus through Cas9/sgRNA-guided specific integration in B-lymphocytes, which was mediated by an integrase-defective lentiviral vector. The edited B cells were capable of differentiating into LLPCs both in vitro and in vivo. Transcriptional profiling analysis confirmed that these cells were typical LLPCs. Importantly, these cells secreted de novo antibodies persistently, which were able to inhibit human melanoma growth via an antibody-mediated checkpoint blockade in xenograft-tumor mice. Our work suggests that the engineered LLPCs may be utilized as a vehicle to constantly produce special antibodies for long-term cellular immunotherapy to eradicate tumors and cellular reservoirs for various pathogens including human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV).