Biophysical and Computational Studies of the vCCI:vMIP-II Complex.

Certain viruses have the ability to subvert the mammalian immune response, including interference in the chemokine system. Poxviruses produce the chemokine binding protein vCCI (viral CC chemokine inhibitor; also called 35K), which tightly binds to CC chemokines. To facilitate the study of vCCI, we first provide a protocol to produce folded vCCI from Escherichia coli (E. coli.) It is shown here that vCCI binds with unusually high affinity to viral Macrophage Inflammatory Protein-II (vMIP-II), a chemokine analog produced by the virus, human herpesvirus 8 (HHV-8). Fluorescence anisotropy was used to investigate the vCCI:vMIP-II complex and shows that vCCI binds to vMIP-II with a higher affinity than most other chemokines, having a Kd of 0.06 ± 0.006 nM. Nuclear magnetic resonance (NMR) chemical shift perturbation experiments indicate that key amino acids used for binding in the complex are similar to those found in previous work. Molecular dynamics were then used to compare the vCCI:vMIP-II complex with the known vCCI:Macrophage Inflammatory Protein-1ß/CC-Chemokine Ligand 4 (MIP-1ß/CCL4) complex. The simulations show key interactions, such as those between E143 and D75 in vCCI/35K and R18 in vMIP-II. Further, in a comparison of 1 µs molecular dynamics (MD) trajectories, vMIP-II shows more overall surface binding to vCCI than does the chemokine MIP-1ß. vMIP-II maintains unique contacts at its N-terminus to vCCI that are not made by MIP-1ß, and vMIP-II also makes more contacts with the vCCI flexible acidic loop (located between the second and third beta strands) than does MIP-1ß. These studies provide evidence for the basis of the tight vCCI:vMIP-II interaction while elucidating the vCCI:MIP-1ß interaction, and allow insight into the structure of proteins that are capable of broadly subverting the mammalian immune system.

Mucosal immunization with Ad5-based vaccines protects Syrian hamsters from challenge with omicron and delta variants of SARS-CoV-2.

SARS-CoV-2 variant clades continue to circumvent antibody responses elicited by vaccination or infection. Current parenteral vaccination strategies reduce illness and hospitalization, yet do not significantly protect against infection by the more recent variants. It is thought that mucosal vaccination strategies may better protect against infection by inducing immunity at the sites of infection, blocking viral transmission more effectively, and significantly inhibiting the evolution of new variants of concern (VOCs). In this study, we evaluated the immunogenicity and efficacy of a mucosally-delivered, non-replicating, adenovirus type 5-vectored vaccine that expresses the spike (S) gene of Wuhan (rAd5-S-Wuhan), delta (rAd5-S-delta), or omicron (rAd5-S-omicron) SARS-CoV-2 VOCs. Hamsters were immunized with these vaccines intranasally prior to challenge with omicron or delta variants. Additionally, one group was vaccinated by oral gavage with rAd5-S-Wuhan prior to challenge with the delta variant. Both intranasal and oral administration of rAd5-S-Wuhan generated cross-reactive serum IgG and mucosal IgA to all variant spike and RBD proteins tested. rAd5-S-omicron and rAd5-S-delta additionally elicited cross-reactive antibodies, though rAd5-S-omicron had significantly lower binding antibody levels except against its matched antigens. Two weeks after the final vaccination, hamsters were challenged with a SARS-CoV-2 variant; omicron or delta. Whether matched to the challenge or with rAd5-S-Wuhan, all vaccines protected hamsters from weight loss and lung pathology caused by challenge and significantly reduced viral shedding compared to placebo. Vaccination with rAd5-S-Wuhan provided significant protection, although there was an improved reduction in shedding and disease pathology in groups protected by the matched VOC vaccines. Nevertheless, Wuhan-based vaccination elicited the most cross-reactive antibody responses generally. Overall, heterologous vaccination via mucosal routes may be advantageous for second-generation vaccines.