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Antisera induced by immunization of rabbits with the synthetic peptide P56/75, which has the amino acid (aa) sequence from aa56 to aa75 of HPV16 L2, neutralize pseudovirions and raft-virions of multiple high-risk HPV types, indicating that cross-neutralization epitopes are present in the aa56-75 region. We generated two mouse monoclonal antibodies (MAb): MAb13B and MAb24B recognizing the regions of aa64-73 and aa58-64, respectively. The neutralization assay using pseudovirions of HPV16, 18, 31, 33, 35, 51, 52 and 58 showed that MAb13B neutralized HPV16, 18, and 51, and MAb24B neutralized all the types tested. The mixture of MAb13B and MAb24B neutralized HPV16, 18, and 51 pseudovirions more efficiently than each of the MAbs alone. The data indicate that there are at least two cross-neutralization epitopes in the aa56-75 region and that an antigen capable of presenting the two cross-neutralization epitopes would be a good vaccine candidate for a broad-spectrum of HPVs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Rabbit anti-HPV16 L2 serum (anti-P56/75) neutralizes multiple oncogenic human papillomaviruses (HPVs). We inoculated HeLa cells with HPV16 pseudovirus (16PV) and with anti-P56/75-bound 16PV (16PV-Ab). Both 16PV and 16PV-Ab attached equally well to the cell surface. However, the cell-attached L1 protein of 16PV became trypsin-resistant after incubation at 37°C, whereas approximately 20% of the cell-attached 16PV-Ab L1 remained trypsin-sensitive. Confocal microscopy of HeLa cells inoculated with 16PV revealed packaged DNA in the nucleus at 22h after inoculation; however, nuclear DNA was not detected in cells inoculated with 16PV-Ab. Electron microscopy of HeLa cells inoculated with 16PV showed particles located in multivesicular bodies, lamellar bodies, and the cytosol after 4h; no cytosolic particles were detected after inoculation with 16PV-Ab. These data suggest that anti-P56/75 inhibits HPV infection partly by blocking viral entry and primarily by blocking the transport of the viral genome to the nucleus., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The deduced amino acid (aa) sequence of L2 of the newly sequenced HPV51 strain, isolated by Matsukura and Sugase (Ma-strain), was markedly different from that of the prototype HPV51 isolated by Nuovo et al. (Nu-strain) (GenBank M62877) in two regions: aa 95-99 (region I) and aa 179-186 (region II). The two regions of Ma-strain were homologous to those of the other mucosal HPVs. The aa sequences of the N-terminal and C-terminal regions of Ma-L2 and Nu-L2 were identical and contained the nuclear localizing signal (NLS). When expressed in HEK293 cells, Ma-strain L2 (Ma-L2) was located in the nucleus but Nu-strain L2 (Nu-L2), in the cytoplasm. The chimeric L2s having both Nu-L2 regions I and II were located in the cytoplasm, and those having one of them were located both in the nucleus and cytoplasm, suggesting that Nu-L2 regions I and II inhibit the NLS function. For a better understanding of a role of L2 in infection, pseudovirion (PV) preparations were produced with a reporter, Ma-strain L1, and various L2s (Ma-L2, Nu-L2, or the chimeric L2s). These PV preparations contained structurally similar particles composed of L1 and L2 and the packaged reporter plasmid at a similar level. The reporter expression was not induced in HEK293 cells after inoculation with PVs containing the L2s that are incapable of localizing in the nucleus when expressed alone. Among PVs containing L2s capable of localizing in the nucleus, the reporter expression was induced only by PVs containing Ma-L2 region I. Thus, the results indicate that the expression of the reporter in the HPV51 PV requires the nuclear localizing ability of L2 and another unknown function associated with region I.

In host animals, adeno-associated virus (AAV) is detectable mainly in the lymphoid tissue, which appears to be a target in natural infection. We used the human monocytic cell lines THP-1 and U937 to study the effect of mouse anti-AAV2 antiserum on infection with an AAV2 vector having the luciferase gene (AAV2/Luc). AAV2/Luc was found to infect THP-1 and U937 cells much less efficiently than HeLa cells, as monitored with the induced enzyme activity. Pre-incubation of AAV2/Luc with anti-AAV2 antiserum at a sub-neutralizing concentration enhanced by 2-to-10 fold infection of THP-1 and U937 with AAV2/Luc, but not of HeLa. Similarly, anti-AAV10 serum at a low level enhanced infection of THP-1 with AAV10/Luc. Sera of two cynomolgus monkeys, which had been probably infected with an AAV2-like virus, enhanced infection of THP-1 with AAV2/Luc. The enhancement was reduced with blocking the IgG-receptors Fcgamma-RI and Fcgamma-RII, which were displayed on the surface of THP-1 and U937 but not HeLa cells, with anti-Fcgamma-RI antibody or anti-Fcgamma-RII antibody. The data indicate that infection of Fcgamma receptor-bearing cells with AAV is enhanced by anti-AAV IgG antibodies at a sub-neutralizing concentration that play a role in linking AAV particles and Fcgamma receptors.

Neutralizing antibody against human papillomavirus (HPV) minor capsid protein L2 can cross-neutralize different HPV genotypes in vitro. To identify the segments containing the cross-neutralization epitopes of HPV16 L2, we characterized antisera obtained by immunizing two rabbits with each of the ten synthetic peptides of 14 to 20 amino acids (aa) long, which represents a part of the HPV16 L2 sequence from aa 14 to 144. The antisera against the peptides within the region from aa 18 to 144 efficiently bound to HPV16 L1/L2-capsids and neutralized HPV16 pseudovirions, indicating that the region is displayed on the surface of the capsids and contains several neutralization epitopes. Antiserum against the peptide from aa 18 to 38 (anti-P18/38) cross-neutralized HPV18. Anti-P56/75 cross-neutralized HPV18, 31, and 58. Anti-P61/75 and anti-P64/81 cross-neutralized HPV18 and 58. Anti-P96/115 and the antiserum induced by a mutant P96/115 (S and T at aa 101 and 112 were replaced with L and S, respectively) cross-neutralized HPV31 and 58. The mixture of equal volumes of three antisera, anti-P18/38, anti-P56/75, and anti-mutant P96/115, neutralized HPV16, 18, 31, and 58 more efficiently than anti-P56/75 alone, suggesting that there is a synergistic effect of antibodies on the cross-neutralization. The cross-neutralization appears to be correlated with conserved aa sequences among HPV types. The data in this study provide a basis for designing vaccine antigens effective against a broader spectrum of the high-risk HPVs.

The P670 promoter of HPV16 directs transcription of the virus late genes in the differentiating epithelium. We found that CCAAT/enhancer binding protein beta (C/EBPbeta), a key transcription factor that induces the terminal differentiation of keratinocytes, enhanced the P670-driven transcription in transient reporter assays in HeLa cells and human primary keratinocytes, whereas it inhibited, as reported previously, the transcription from the early P97 promoter. An electrophoretic mobility shift analysis identified two binding sites in the upstream region of P670 for a bacterially expressed C/EBPbeta. A chromatin immunoprecipitation analysis demonstrated that C/EBPbeta bound to these sites of the P670 reporter plasmid in HeLa cells. Nucleotide substitutions in these sites in the reporter plasmid abrogated the enhancement by C/EBPbeta in the transient HeLa and keratinocyte assays, indicating that the C/EBPbeta-binding to these sites is required for the enhancement of transcription from P670. These results suggest that C/EBPbeta is involved in enhancing transcription from the P670 during keratinocyte differentiation.

We demonstrated the presence of two adeno-associated viruses (AAVs), designated AAV10 and AAV11, in cynomolgus monkeys by isolating and sequencing the entire viral coding regions from the monkey DNA. AAV10 and AAV11 capsid proteins shared 84% and 65%, respectively, of amino acids with AAV2. A phylogenetic analysis of AAV capsid proteins showed that AAV10 and AAV11 resembled most AAV8 and AAV4, respectively. To characterize the capsid protein, we pseudotyped an AAV2 vector with the monkey AAV capsid proteins and examined the resulting pseudotypes AAV2/10 and AAV2/11, in comparison with the AAV2 vector, for their host ranges in cell lines and tissue tropism in mice. AAV2/10 and AAV2/11 transduced primate cells less efficiently than AAV2. Whereas AAV2 transduced undifferentiated C2C12 mouse myoblasts more efficiently than differentiated ones, AAV2/10 and AAV2/11 transduced the undifferentiated myoblasts less efficiently than differentiated ones. Three weeks after injection to the muscle of the hind legs, AAV2/10 and AAV2 induced transgene expression similarly, but AAV2/11 did not transduce the skeletal muscle. Six weeks after systemic administration, transduced vector DNA was detected by PCR in the liver and spleen of mice inoculated with AAV2, in the liver, heart, muscle, lung, kidney, and uterus of mice with AAV2/10, and the muscle, kidney, spleen, lung, heart, and stomach of mice with AAV2/11. Mouse antisera against capsid protein VP2 of the three AAVs neutralized the respective vector particles in a type-specific manner. The results indicate that AAV10 and AAV11 capsid proteins, which are antigenically distinct from each other and AAV2, are likely to determine their host ranges and tissue tropism that are different from AAV2s, suggesting that cynomolgus AAVs could provide a broader choice of pseudotype AAV vectors for gene therapy.

Human papillomavirus 16 major capsid protein L1 (composed of 505 amino acids (aa) including 12 cysteines) assembles by itself into virion-like icosahedral particles (L1-capsids), each of which is dissociated into 72 pentameric capsomeres when intermolecular disulfide bonds are disrupted. To identify the cysteines affecting the bonding and the structural integrity of the L1-capsids, we constructed a series of L1 mutants with substitution of serine for cysteine, which were expressed from recombinant baculoviruses in the insect Sf9 cells. From infected cells, the self-assembled L1-capsid fractions were purified by CsCl-equilibrium centrifugation and examined for velocity sedimentation profiles, for the presence of intermolecular bonding by SDS-PAGE with or without a reducing agent, for morphology under an electron microscope, and for susceptibility to trypsin digestion. Mutants C175S (C at aa 175 was replaced with S) and C185S were sedimented in sucrose-density gradients slightly slower than the wild type (WT) capsids, and mutant C428S stayed near the top as WT-capsomeres did. In the nonreducing SDS gel, where WT-capsids were separated into two bands of L1-trimers and L1-dimers, the C175S-trimer band was not detected, the C185S-dimer band was much less dense, and the C428S-trimer and C428S-dimer bands were not detected. Thus, it seems likely that C175, C185, and C428 are involved in L1 trimerization, in L1 dimerization, and in both, respectively. Morphologically, the C175S, C185S, and C428S fractions appeared to consist mostly of heterogeneous rod-shaped tubules, of smaller spherical particles, and of only capsomeres, respectively, whereas C102S, C229S, and C379S resembled WT. The C161S, C175S, C185S, C229S, C379S, and C428S capsids were more sensitive to degradation caused by trypsin than WT. The results indicate that C175, C185, and C428 are required for the normal assembly of L1-capsids through trimerization and dimerization of L1 bound by the intercapsomeric disulfide bonds between cysteines, and that C161, C229, and C379 are necessary for the integrity of L1-capsids probably through intramolecular bonding.

Adenoassociated virus (AAV) provirus in latently infected cells is rescued by superinfection with adenovirus. We examined helper-independent rescue of AAV by Fas-mediated apoptosis from the HeLa lines with AAV DNA integrated in chromosome (HeLa/AAV). In HeLa/AAV, anti-Fas antibody was found to induce low-level production of AAV virions, as detected by the presence of AAV DNA protected from DNase digestion. The antibody induced higher virion production in Fas-enriched HeLa/AAV, which was generated by transfecting HeLa/AAV with an expression plasmid for Fas, than in HeLa/AAV, and the rescued virions were shown to be infectious as assayed along with adenovirus. The antibody also induced apoptotic DNA fragmentation, as detected by staining intracellular fragmented DNA and electrophoresis, in HeLa/AAV and, more markedly, in Fas-enriched HeLa/AAV. Furthermore, an inhibitor for caspase-8 suppressed both the AAV virion production and the DNA fragmentation. Thus, the integrated AAV DNA is likely to be induced to initiate a low level of replication when Fas-mediated apoptosis is activated in HeLa cells.