Gregory C. Wipf

Identification of an overprinting gene in Merkel cell polyomavirus provides evolutionary insight into the birth of viral genes

Many viruses use overprinting (alternate reading frame utilization) as a means to increase protein diversity in genomes severely constrained by size. However, the evolutionary steps that facilitate the de novo generation of a novel protein within an ancestral ORF have remained poorly characterized. Here, we describe the identification of an overprinting gene, expressed from an Alternate frame of the Large T Open reading frame (ALTO) in the early region of Merkel cell polyomavirus (MCPyV), the causative agent of most Merkel cell carcinomas. ALTO is expressed during, but not required for, replication of the MCPyV genome. Phylogenetic analysis reveals that ALTO is evolutionarily related to the middle T antigen of murine polyomavirus despite almost no sequence similarity. ALTO/MT arose de novo by overprinting of the second exon of T antigen in the common ancestor of a large clade of mammalian polyomaviruses. Taking advantage of the low evolutionary divergence and diverse sampling of polyomaviruses, we propose evolutionary transitions that likely gave birth to this protein. We suggest that two highly constrained regions of the large T antigen ORF provided a start codon and C-terminal hydrophobic motif necessary for cellular localization of ALTO. These two key features, together with stochastic erasure of intervening stop codons, resulted in a unique protein-coding capacity that has been preserved ever since its birth. Our study not only reveals a previously undefined protein encoded by several polyomaviruses including MCPyV, but also provides insight into de novo protein evolution.

Immunization of mice with HPV vaccinia virus recombinants generates serum IgG, IgM,and mucosal IgA antibodies

To assess the utility of vaccinia virus recombinants in the development of an immune response against HPV capsid antigens, 5-week-old C57B16 female mice were administered either purified HPV 1 capsids produced by a vaccinia virus recombinant or the recombinant vaccinia virus itself. Animals were boosted at Week 4 with either agent. Mice developed a serum IgG antibody response in all the administration protocols that was directed mainly against native L1 epitopes. Mice injected initially with the vaccinia virus recombinant and boosted with purified capsids had a higher titer antibody response (P = 0.024) with more mice responding to a greater extent. All mice produced a serum IgM response that preceded the IgG response by approximately 2 weeks and lasted 1-3 weeks. The IgM response was directed against native L1 epitopes. Although no serum IgA was detected, IgA could be detected in vaginal secretions of mice that were immunized or boosted with the vaccinia virus vector. These results indicate that an extensive humoral immune response to HPV can be elicited using vaccinia virus recombinants.

A Single Human Papillomavirus Vaccine Dose Improves B Cell Memory in Previously Infected Subjects

Although licensed human papillomavirus (HPV) vaccines are most efficacious in persons never infected with HPV, they also reduce infection and disease in previously infected subjects, indicating natural immunity is not entirely protective against HPV re-infection. The aim of this exploratory study was to examine the B cell memory elicited by HPV infection and evaluate whether vaccination merely boosts antibody (Ab) levels in previously infected subjects or also improves the quality of B cell memory. Toward this end, the memory B cells (Bmem) of five unvaccinated, HPV-seropositive subjects were isolated and characterized, and subject recall responses to a single HPV vaccine dose were analyzed. Vaccination boosted Ab levels 24- to 930-fold (median 77-fold) and Bmem numbers 3- to 27-fold (median 6-fold). In addition, Abs cloned from naturally elicited Bmem were generally non-neutralizing, whereas all those isolated following vaccination were neutralizing. Moreover, Ab and plasmablast responses indicative of memory recall responses were only observed in two subjects. These results suggest HPV vaccination augments both the magnitude and quality of natural immunity and demonstrate that sexually active persons could also benefit from HPV vaccination. This study may have important public policy implications, especially for the older ‘catch-up’ group within the vaccines target population.

Risk of squamous cell skin cancer after organ transplant associated with antibodies to cutaneous papillomaviruses, polyomaviruses, and TMC6/8 (EVER1/2) variants.

Squamous cell skin cancer (SCSC) disproportionately affects organ transplant recipients, and may be related to increased viral replication in the setting of immune suppression. We conducted a nested case–control study among transplant recipients to determine whether SCSC is associated with antibodies to cutaneous human papillomaviruses (HPV), to genes associated with a rare genetic susceptibility to HPV (TMC6/TMC8), or to human polyomaviruses (HPyV). Cases (n = 149) had histologically confirmed SCSC, and controls (n = 290) were individually matched to cases on time since transplant, type of transplant, gender, and race. All subjects had serum drawn immediately prior to transplant surgery. Antibodies to 25 cutaneous HPVs and six HPyVs were assayed by detection of binding to virus‐like particles, and 11 TMC6/8 variants were genotyped. After correction for multiple comparisons, only antibodies to HPV37 were associated with SCSC (OR 2.0, 95% CI 1.2–3.4). Common genetic variants of TMC6/8 were not associated with SCSC, but three variants in TMC8 (rs12452890, rs412611, and rs7208422) were associated with greater seropositivity for species 2 betapapillomaviruses among controls. This study suggests that some betaHPVs, but not polyomaviruses, may play a role in the excess risk of SCSC among transplant recipients.

Analysis of Memory B-Cell Responses Reveals Suboptimal Dosing Schedule of a Licensed Vaccine

High levels of human papillomavirus vaccine–elicited neutralizing serum antibodies elicited to the first and second doses strongly correlated with reduced memory B-cell responses to the third dose, including antibody responses.