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To address how L2-specific antibodies prevent human papillomavirus (HPV) infection of the genital tract, we generated neutralizing monoclonal antibodies (MAbs) WW1, a rat IgG2a that binds L2 residues 17 to 36 (like mouse MAb RG1), and JWW3, a mouse IgG2b derivative of Mab24 specific for L2 residues 58 to 64. By Western blotting, WW1 recognized L2 of 29/34 HPV genotypes tested, compared to only 13/34 for RG1 and 25/34 for JWW3. WW1 IgG and F(ab')2 bound HPV16 pseudovirions similarly; however, whole IgG provided better protection against HPV vaginal challenge. Passive transfer of WW1 IgG was similarly protective in wild-type and neonatal Fc receptor (FcRn)-deficient mice, suggesting that protection by WW1 IgG is not mediated by FcRn-dependent transcytosis. Rather, local epithelial disruption, required for genital infection and induced by either brushing or nonoxynol-9 treatment, released serum IgG in the genital tract, suggesting Fc-independent exudation. Depletion of neutrophils and macrophages reduced protection of mice upon passive transfer of whole WW1 or JWW3 IgGs. Similarly, IgG-mediated protection by L2 MAbs WW1, JWW3, and RG1 was reduced in Fc receptor knockout compared to wild-type mice. However, levels of in vitro neutralization by WW1 IgG were similar in TRIM21 knockout and wild-type cells, indicating that Fc does not contribute to antibody-dependent intracellular neutralization (ADIN). In conclusion, the Fc domain of L2-specific IgGs is not active for ADIN, but it opsonizes bound extracellular pseudovirions for phagocytes in protecting mice from intravaginal HPV challenge. Systemically administered neutralizing IgG can access the site of infection in an abrasion via exudation without the need for FcRn-mediated transcytosis.IMPORTANCE At least 15 alpha HPV types are causative agents for 5% of all cancers worldwide, and beta types have been implicated in nonmelanoma skin cancer, whereas others produce benign papillomas, such as genital warts, associated with considerable morbidity and health systems costs. Vaccines targeting the minor capsid protein L2 have the potential to provide broad-spectrum immunity against medically relevant HPVs of divergent genera via the induction of broadly cross-neutralizing serum IgG. Here we examine the mechanisms by which L2-specific serum IgG reaches the viral inoculum in the genital tract to effect protection. Abrasion of the vaginal epithelium allows the virus to access and infect basal keratinocytes, and our findings suggest that this also permits the local exudation of neutralizing IgG and vaccine-induced sterilizing immunity. We also demonstrate the importance of Fc-mediated phagocytosis of L2 antibody-virion complexes for humoral immunity, a protective mechanism that is not detected by current in vitro neutralization assays.

A high rate of genetic and antigenic variability among porcine reproductive and respiratory syndrome viruses (PRRSVs) hampers effective prevention and control of the disease caused by PRRSV. The major envelope protein (GP5) encoded by the ORF5 of PRRSV has a critical role in inducing virus neutralizing (VN) antibody and cross protection among different strains of PRRSV. This study was conducted to identify sequence elements related to cross neutralization by comparing the ORF5 sequences of 69 field isolates in conjunction with their susceptibility to VN antibody raised against the VR2332 strain in vitro and in vivo . Five common variable sites (amino acid position 32–34, 38–39, 57–59, 137 and 151) were identified between susceptible and resistant viral isolates. Mutants whose ORF5 amino acid sequences were substituted with the sequences corresponding to the 5 identified common variable sites individually or concurrently were generated from a VR2332-backboned infectious clone by site mutagenesis. The change in the susceptibility of the mutants to VN antibodies specific for VR2332 or a heterologous PRRSV was assessed to determine the association of those 5 identified sites with cross neutralization. Among the five sites, the changes of amino acid sequences at three sites (32–34, 38–39, and 57–59) located in the N-terminal ectodomain of ORF5 significantly influenced the susceptibility of the mutant viruses to VN antibody, suggesting that sequence homology at these sites can be utilized as genetic markers to predict the degree of cross neutralization among different PRRSVs.

The influenza A virus M2 protein has important roles during virus entry and in the assembly of infectious virus particles. The cytoplasmic tail of the protein can be palmitoylated at a cysteine residue, but this residue is not conserved in a number of human influenza A virus isolates. Recombinant viruses encoding M2 proteins with a serine substituted for the cysteine at position 50 were generated in the A/WSN/33 (H1N1) and A/Udorn/72 (H3N2) genetic backgrounds. The recombinant viruses were not attenuated for replication in MDCK cells, Calu-3 cells, or in primary differentiated murine trachea epithelial cell cultures, indicating there was no significant contribution of M2 palmitoylation to virus replication in vitro. The A/WSN/33 M2C50S virus displayed a slightly reduced virulence after infection of mice, suggesting that there may be novel functions for M2 palmitoylation during in vivo infection.

An automated PCR with fluorescent probes (molecular beacons) detected Mycobacterium avium subsp. paratuberculosis in bovine feces. When the PCR was compared with culture in testing 41 fecal samples, kappa scores of 0.94 to 0.96, a sensitivity of 93 to 96%, and a specificity of 92% were obtained. Results were quantitated by using a standard curve derived from a plasmid containing IS 900 . A minimum quantity of 1.7 × 10 −4 pg of DNA, correlating to 1 to 8 CFU, was detected.

The origin recognition complex (ORC) coordinates a series of events that lead to initiation of DNA strand duplication. As a nuclear double stranded DNA plasmid, the papillomavirus (PV) genome resembles a mini-chromosome in infected cells. To initiate its replication, the viral E2 protein binds to and recruits the E1 DNA helicase at the viral origin. PV genome replication program exhibits three stages: initial amplification from a single genome upon infection to a few copies per cell, a cell cycle linked maintenance phase, and a differentiation dependent late stage where the genome is amplified to thousands of copies. Involvement of ORC or other pre-replication complex (pre-RC) factors has not been described. We report that human PV (HPV) and bovine PV (BPV-1) E2 proteins bind to ORC2, however, ORC2 was not detected at the viral origin. Depletion of ORC2 enhanced PV replication in a transient replication model and in keratinocytes stably maintaining viral episomes, while there was no effect on copy number in a cell line with integrated HPV genomes. Consistent with this, occupancy of E1 and E2 at the viral origin increased following ORC2 silencing. These data imply that ORC2 is not necessary for activation of the PV origin by E1 and E2 but instead suppresses E2 replicative function. Furthermore, we observed that over-expression of HPV E2 decreased ORC2 occupation at two known mammalian origins of replication, suggesting that E2 restricts pre-ORC assembly that could otherwise compete for host replication complexes necessary for viral genome amplification. We infer that the ORC2 complex with E2 restricts viral replication in the maintenance phase of the viral replication program and that elevated levels of E2 that occur during the differentiation dependent amplification stage subvert ORC loading and hence DNA synthesis at cellular origins., Author Summary Papillomavirus genome replication occurs during three distinct stages that are linked to the differentiation state of the infected epithelium. The viral proteins E1 and E2 recognize the viral origin and initiate a process that attracts host DNA replication factors. The origin recognition complex (ORC) coordinates initiation of chromosome duplication. While ORC2 binds to the E2 protein, its depletion does not impair PV genome replication. Instead, depletion of ORC2 stimulates viral replication, while over-expression of E2 protein decreases ORC2 occupancy at mammalian origins. We propose that the relative abundance of E2 and ORC2 in complex regulates viral and cellular origin licensing.

The use of live recombinant attenuated Salmonella vaccines (RASV) is a promising approach for controlling infections by multiple pathogens. The highly conserved extracellular domain of the influenza M2 protein (M2e) has been shown to provide broad spectrum protection against multiple influenza subtypes sharing similar M2e sequences. An M2e epitope common to a number of avian influenza subtypes was inserted into the core antigen of woodchuck hepatitis virus and expressed in two different recombinant attenuated Salmonella Typhimurium strains. One strain was attenuated via deletion of the cya and crp genes. The second strain was engineered to exhibit a programmed delayed lysis phenotype. Both strains were able to produce both monomeric fusion proteins and fully assembled core particles. Mice orally immunized with the strain exhibiting delayed lysis induced significantly greater antibody titers than the Δcya Δcrp strain and provided moderate protection against weight loss to a low level challenge with the influenza strain A/WSN/33 modified to express the M2e sequence common to avian viruses. Further studies indicated that the Salmonella expressed core antigen induced comparable antibody levels to the purified core antigen injected with an alum adjuvant and that both are able to reduce viral replication in the lungs. To our knowledge this is the first report demonstrating Salmonella-mediated delivery of influenza virus M2e protein in a mammalian host to induce a protective immune response against viral challenge.

A carboxy-terminal epitope tag introduced into the coding region of the A/WSN/33 M2 protein resulted in a recombinant virus (rWSN M2myc) which replicated to titers similar to those of the parental virus (rWSN) in MDCK cells. The rWSN M2myc virus was attenuated in its ability to induce mortality and weight loss after the intranasal inoculation of BALB/c mice, indicating that the M2 cytoplasmic tail plays a role in virus virulence. Mice infected with rWSN M2myc were completely protected from subsequent challenge with rWSN, suggesting that epitope tagging of the M2 protein may be a useful way of attenuating influenza A virus strains.

Swine influenza viruses (SIV) naturally infect pigs and can be transmitted to humans. In the pig, genetic reassortment to create novel influenza subtypes by mixing avian, human, and swine influenza viruses is possible. An SIV vaccine inducing cross-protective immunity between different subtypes and strains circulating in pigs is highly desirable. Previously, we have shown that an H3N2 SIV (A/swine/Texas/4199-2/98 [TX98]) containing a deleted NS1 gene expressing a truncated NS1 protein of 126 amino acids, NS1Œ126, was attenuated in swine. In this study, 4-week-old pigs were vaccinated with the TX98 NS1Œ126 modified live virus (MLV). Ten days after boosting, pigs were challenged with wild-type homologous H3N2 or heterosubtypic H1N1 SIV and sacrificed 5 days later. The MLV was highly attenuated and completely protected against challenge with the homologous virus. Vaccinated pigs challenged with the heterosubtypic H1N1 virus demonstrated macroscopic lung lesions similar to those of the unvaccinated H1N1 control pigs. Remarkably, vaccinated pigs challenged with the H1N1 SIV had significantly lower microscopic lung lesions and less virus shedding from the respiratory tract than did unvaccinated, H1N1-challenged pigs. All vaccinated pigs developed significant levels of hemagglutination inhibition and enzyme-linked immunosorbent assay titers in serum and mucosal immunoglobulin A antibodies against H3N2 SIV antigens. Vaccinated pigs were seronegative for NS1, indicating the potential use of the TX98 NS1Œ126 MLV as a vaccine to differentiate infected from vaccinated animals. Swine influenza virus (SIV) is an important swine pathogen involved in the porcine respiratory disease complex in most swine-producing countries. Serological and virological studies demonstrated 23 to 28% prevalence of SIV in swine populations in the midwestern and north central United States (4, 35, 56). Mortality of SIV in pigs is low, while morbidity may approach 100%. SIV-affected pigs show clinical signs of anorexia and weight loss, fever, respiratory distress, coughing, and nasal discharge (18). SIVs currently circulating in North American swine are subtypes H1N1, H3N2, and H1N2 (36). From 1930 to 1998, classical H1N1 viruses were the subtype predominantly isolated from U.S. swine. In 1998, a new SIV subtype, H3N2, emerged from reassortment of swine, human, and avian influenza viruses (19, 56, 58). The H3N2 SIV acquired PB1, hemagglutinin (HA), and NA genes from recent human viruses, PB2 and PA from avian viruses, and NP, M, and NS genes from the classical H1N1 swine virus (19, 56, 58). A year

Capsomers were produced in bacteria as glutathione-S-transferase (GST) fusion proteins with human papillomavirus type 16 L1 lacking the first nine and final 29 residues (GST-HPV16L1Δ) alone or linked with residues 13-47 of HPV18, HPV31 and HPV45 L2 in tandem (GST-HPV16L1Δ-L2x3). Subcutaneous immunization of mice with GST-HPV16L1Δ or GST-HPV16L1Δ-L2x3 in alum and monophosphoryl lipid A induced similarly high titers of HPV16 neutralizing antibodies. GST-HPV16L1Δ-L2x3 also elicited moderate L2-specific antibody titers. Intravaginal challenge studies showed that immunization of mice with GST-HPV16 L1Δ or GST-HPV16L1Δ-L2x3 capsomers, like Cervarix®, provided complete protection against HPV16. Conversely, vaccination with GST-HPV16 L1Δ capsomers failed to protect against HPV18 challenge, whereas mice immunized with either GST-HPV16L1Δ-L2x3 capsomers or Cervarix® were each completely protected. Thus, while the L2-specific response was moderate, it did not interfere with immunity to L1 in the context of GST-HPV16L1Δ-L2x3 and is sufficient to mediate L2-dependent protection against an experimental vaginal challenge with HPV18.