Despite the extensive molecular information on serum-derived human hepatitis B viruses (HBV), liver-derived replicative HBV genomes have remained largely uninvestigated. We have examined the sequences of the entire core antigen (nucleocapsid) of liver-derived HBVs in 15 different hepatoma patients. Bona fide mutations, rather than subtype polymorphism, have been identified based on the high-frequency occurrence of structural differences from wild type at the highly evolutionarily conserved positions, instead of at the positions known to contain genetic heterogeneity among different isolates from different geographic locations. The distribution of these naturally occurring mutations of HBV core gene appears to be nonrandom and is found predominantly within three major (I, IV, and V) and four minor domains (II, III, VI, and VII). In general, domain IV mutations correlate with domain V mutations. The replicative HBV DNAs tend to accumulate a higher number of mutated core domains than the integrated HBV DNAs. At the domain level, there is no significant difference in HBV core mutation frequencies between the liver tumors and the adjacent nontumorous livers. Strikingly, domains I, III, and V coincide with three major known T cell epitopes within the core protein in acute and chronic hepatitis B patients. Furthermore, these domains coincide with HLA class II-restricted T cell epitopes, rather than with the conventional HLA class I-restricted epitopes of cytotoxic T lymphocytes. Our results support the hypothesis that HBV core antigen variants can accomplish immunoevasion via accumulated escape mutations. In addition, they also provide a potential molecular explanation for the maintenance of persistent infection of human hepatitis B virus in chronic carriers.

We developed a severe acute respiratory syndrome (SARS) subunit recombinant protein vaccine candidate based on a highyielding, yeast-engineered, receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel ® , RBD219-N1 induced high-level neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel ® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ~ 30% mortality in mice when immunized with the SARS S protein formulated with Alhydrogel ® , and 100% mortality in negative controls. An RBD219-N1 formulation Alhydrogel ® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge.Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel ® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and other emerging and re-emerging beta-CoVs such as SARS-CoV-2.

Studying the mechanisms of host survival resulting from viral encephalitis is critical to the development of vaccines. Here we have shown in several independent studies that high-dose treatment with neutralizing antibody prior to intranasal infection with Venezuelan equine encephalitis virus had an antiviral effect in the visceral organs and prolonged survival time of infected mice, even in the absence of alpha beta T cells. Nevertheless, the antibody treatment did not prevent the development of lethal encephalitis. In contrary, the adoptive transfer of primed CD4 + T cells is necessary to prevent lethal encephalitis in mice lacking alpha beta T cell receptor.

The elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), a mosquito-borne flavivirus, has induced severe neurological symptoms, mostly in the elderly population. No vaccines are available for human use. Recent work showed that an attenuated WNV, a nonstructural (NS) 4B-P38G mutant, induced no lethality but strong immune responses in young (6-to 10-week-old) mice. While studying protective efficacy, we found unexpectedly that old (21-to 22-month) mice were susceptible to WNV NS4B-P38G mutant infection but were protected from subsequent lethal wild-type WNV challenge. Compared to responses in young mice, the NS4B-P38G mutant triggered higher inflammatory cytokine and interleukin-10 (IL-10) production, a delayed ␥␦ T cell expansion, and lower antibody and WNVspecific T cell responses in old mice. Toll-like receptor 7 (TLR7) is expressed on multiple types of cells. Impaired TLR7 signaling in old mice led to dendritic cell (DC) antigen-presenting function compromise and a reduced ␥␦ T cell and regulatory T cell (Treg) expansion during NS4B-P38G mutant infection. R848, a TLR7 agonist, decreased host vulnerability in NS4B-P38G-infected old mice by enhancing ␥␦ T cell and Treg expansion and the antigen-presenting capacity of DCs, thereby promoting T cell responses. In summary, our results suggest that dysregulation of TLR7 partially contributes to impaired innate and adaptive T cell responses and an enhanced vulnerability in old mice during WNV NS4B-P38G mutant infection. R848 increases the safety and efficacy during immunization of old mice with the WNV NS4B-P38G mutant. IMPORTANCEThe elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), an emerging mosquito-borne flavivirus, has induced severe neurological symptoms more frequently in the elderly population. No vaccines are available for human use. Here, we used an aged mouse model to investigate the protective efficacy of an attenuated WNV, the nonstructural 4B-P38G mutant, which was previously shown to induce no lethality but strong immune responses in young adult mice. Studies that contribute to a mechanistic understanding of immune defects in the elderly will allow the development of strategies to improve responses to infectious diseases and to increase vaccine efficacy and safety in aging individuals.