Conjugation of a self-antigen to papillomavirus-like particles allows for efficient induction of protective autoantibodies.

High avidity and long-lasting autoantibodies to a self-polypeptide (TNF-alpha) were generated after parenteral vaccination of mice with low doses of virus-like particle-based (VLP-based) vaccines that were constructed by linking mouse TNF-alpha peptides to the surface of papillomavirus VLPs. High-titer autoantibodies were induced with or without coadministration of potent conventional adjuvants, but were enhanced by coadministration of CFA. Compared with immunization with the fusion protein alone, attachment to VLPs increased autoantibody titers 1,000-fold. A comparison of Ab responses against the self (TNF-alpha) and foreign components of the fusion protein showed that VLP conjugation abrogated the ability of the humoral immune system to distinguish between self and foreign. Similar levels of IgM were detected to self and foreign epitopes regardless of the assembly state of the antigen, suggesting that conjugation of self-peptides to VLPs promotes survival or expansion of mature autoreactive B cells. In a mouse model, vaccination with conjugated particles inhibited development of type II collagen-induced arthritis. Together, these results suggest a potentially flexible method to efficiently generate autoantibodies against specific self-proteins that mediate arthritis and other diseases.

High-risk human papillomavirus is sexually transmitted: evidence from a follow-up study of virgins starting sexual activity (intercourse).

Genital human papillomavirus (HPV) infection is generally considered to be sexually transmitted. However, nonsexual spread of the virus has also been suggested. The goal of this study was to assess: (a) the role of sexual intercourse in the transmission of HPV; (b) the determinants for seroconversion; and (c) the correlation between HPV DNA, abnormal cervical cytology, and serological response to HPV16. One hundred virgins and 105 monogamous women were randomly selected from a population-based cohort study in Copenhagen, Denmark, in which the women were examined twice with 2-year interval (interview, cervical swabs, Pap smear, blood samples). The presence of HPV DNA was determined by GP5+/6+ primers based HPV-PCR-EIA. HPV 16 virus-like particles (VLP) antibodies were detected by ELISA. All of the virgins were both HPV DNA negative and seronegative to VLP16, except for one woman who was weakly HPV 6 DNA positive. Only those virgins who initiated sexual activity became HPV DNA positive and/or VLP16 positive. The most important determinant of HPV DNA acquisition was the number of partners between the two examinations. The only significant risk factor for HPV 16 VLP seroconversion among women acquiring HPV DNA was HPV type. Our results show that sexual intercourse is important in the transmission of HPV, and that HPV 16 VLP seroconversion and the development of cervical lesions only occur after HPV transmission. Remarkably, no cervical lesions were found in HPV 16 DNA positive women who had seroconverted. Although based on small numbers, this may suggest that the development of antibodies had a protective effect.

Induction of autoantibodies to mouse CCR5 with recombinant papillomavirus particles.

The vertebrate immune system has evolved to respond vigorously to microbial infection but to ignore self-antigens. Evidence has emerged that B cell responses to viruses are initiated by immune recognition of ordered arrays of antigen on the viral surface. To test whether autoantibodies against a self-antigen can be induced by placing it in a context that mimics the ordered surface of a viral particle, a peptide representing an extracellular loop of the mouse chemokine receptor CCR5 was incorporated into an immunodominant site of the bovine papillomavirus virus L1 coat protein, which self-assembles into virus-like particles. Mice inoculated with chimeric L1-CCR5 particles generated autoantibodies that bound to native mouse CCR5, inhibited binding of its ligand RANTES, and blocked HIV-1 infection of an indicator cell line expressing a human-mouse CCR5 chimera. These results suggest a general method for inducing autoantibodies against self-antigens, with diverse potential basic research and clinical applications.

Changes in the extracellular envelope glycoprotein of variants that evolve during the course of simian immunodeficiency virus SIVMne infection affect neutralizing antibody recognition, syncytium formation, and macrophage tropism but not replication, cytopathicity, or CCR-5 coreceptor recognition.

Simian immunodeficiency virus SIVMne, like human immunodeficiency virus, evolves from a macrophage-tropic, non-syncytium-inducing virus at early times in infection to a T-cell-tropic, syncytium-inducing, cytopathic virus population over the course of progression to AIDS. Because the viruses isolated late in SIVMne infection of macaques include a complex mixture of variants, the viral determinants of such phenotypic changes have not been defined. To identify genetic changes that are important to virus evolution in the host, we constructed chimeric viruses by introducing variant envelope genes representative of proviruses throughout the course of infection and disease into the SIVMne parental clone (SIVMneCL8) that infected the macaque. The chimeric viruses expressed sequences encoding the surface unit of the envelope glycoprotein (Env-SU) of variants cloned between 35 and 170 weeks postinfection. The chimera with Env-SU from 35 weeks postinfection encoded only four changes in V1 compared to SIVMneCL8, whereas the chimeras encoding Env-SU from variants isolated later in infection encoded progressively more mutations both in V1 and elsewhere. Like SIVMneCL8, the chimeras were infectious for CEMx174 cells and macaque peripheral blood mononuclear cells. However, in contrast to SIVMneCL8, the chimeric viruses did not infect macaque macrophages, although each retained the ability to recognize the CCR-5 coreceptor. Thus, these data provide direct evidence that changes which evolve in Env-SU during the course of SIVMne infection do not alter CCR-5 interactions. Viruses encoding Env-SU from the latest times in infection (121 to 170 weeks postinfection), after disease was apparent, were syncytium inducing. However, these viruses were not highly cytopathic, suggesting that additional viral determinants may be required for the rapidly replicating, cytopathic phenotype of the uncloned mixed variant population. Changes in Env-SU did allow the virus to escape serum neutralizing antibodies that recognized the SIVMneCL8 parent. Moreover, the chimera encoding the Env-SU of a virus from 35 weeks postinfection, which differed from SIVMneCL8 only in V1, was not sensitive to neutralization by infected macaque sera, suggesting that V1 may define a portion of the principal neutralizing determinant for SIVMne. Together, these data suggest that SIV variants with changes in the Env-SU may be selected primarily by virtue of their ability to escape neutralizing antibody recognition.

Specific N-linked and O-linked glycosylation modifications in the envelope V1 domain of simian immunodeficiency virus variants that evolve in the host alter recognition by neutralizing antibodies.

During progression to AIDS in simian immunodeficiency virus (SIV) Mne-infected macaques, viral variants are selected that encode sequences with serine and threonine changes in variable region 1 (V1) of the surface component of the viral envelope protein (Env-SU). Because these serine and threonine amino acid changes are characteristic of sites for O-linked and N-linked glycosylation, we examined whether they were targets for modification by carbohydrates. For this purpose, we used several biochemical methods for analyzing the Env-SU protein encoded by chimeras of SIVMneCL8 and envelope sequences cloned from an SIVMneCL8-infected Macaca nemestrina during clinical latency and just after the onset of AIDS. The addition of an N-linked glycan was demonstrated by changes in the electrophoretic mobility of Env-SU, and this was verified by specific glycanase digestions and a detailed analysis of the molecular mass of partially purified Env-SU by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Molecular mass calculations by MALDI-TOF MS also demonstrated an increased mass, from 102.3 to 103.5 kDa, associated with serine and threonine residues predicted to be O-linked glycosylation sites. Together, these data provide the first direct evidence that the carbohydrate profile of Env-SU is distinct in SIV variants that evolve during infection of the host. Moreover, our studies show that these changes in glycosylation in V1 were directly associated with changes in antigenicity. Specifically, serine and threonine changes in V1 allowed the virus to escape neutralization by macaque sera that contained antibodies that could neutralize the parental virus, SIVMneCL8. The escape from antibody recognition appeared to be influenced by either O-linked or N-linked carbohydrate additions in V1. Moreover, when glycine residues were engineered at the positions where serine and threonine changes evolve in V1 of SIVMneCL8, there was no change in antigenicity compared to SIVMneCL8. This suggests that the amino acids in V1 are not part of the linear epitope recognized by neutralizing antibody. More likely, V1-associated carbohydrates mask the major neutralizing epitope of SIV. These experiments indicate that the selection of novel glycosylation sites in the V1 region of envelope during the course of disease is driven by humoral immune responses.

Human immunodeficiency virus type 1 coreceptors participate in postentry stages in the virus replication cycle and function in simian immunodeficiency virus infection.

Primate lentiviruses use chemokine coreceptors in addition to the CD4 receptor to initiate virus infection. Simian immunodeficiency virus (SIV) productively infects human cells expressing CD4 and the human allele of the chemokine coreceptor CCR-5 as efficiently as it infects macaque cells expressing human CD4, suggesting that SIV can function with either a simian or a human coreceptor in conjunction with human CD4. In the same macaque cells expressing human CD4, the replication of human immunodeficiency virus type 1 (HIV-1) is blocked at several stages of infection; some isolates are restricted prior to reverse transcription, while others, including some macrophage-tropic and primary isolates, are restricted at a step after reverse transcription but prior to migration of the preintegration complex to the nucleus. Both blocks in HIV-1 replication can be relieved by either expression of the appropriate human coreceptor (CCR-5 or CXCR-4) or expression of SIV gene products in cis with the HIV-1 envelope as a chimera between SIV and HIV-1 (SHIV). Thus, a virus with a SIV core and HIV-1 envelope can efficiently infect macaque cells expressing human CD4, presumably by interacting with the simian coreceptor, whereas a virus with an HIV-1 core and an HIV-1 envelope requires expression of the human allele of the coreceptor for productive infection of these cells. These studies suggest that there are interactions among the coreceptor, the viral envelope, and another viral gene product that govern postentry steps of virus replication. These data are consistent with the hypothesis that such interactions may be required for translocation of the virus core to the nucleus. Moreover, the differential abilities of SIV and HIV-1 to function in these processes with heterologous primate coreceptors may have implications for cross-species transmission.

Characterization of a CD4-expressing macaque cell line that can detect virus after a single replication cycle and can be infected by diverse simian immunodeficiency virus isolates.

Primate lentiviruses such as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) are phenotypically diverse, and virus isolates vary in cytopathicity, replication rate, and cell tropism. While all virus isolates infect primary peripheral blood lymphocytes, only a subset of strains infect established CD4-expressing T-cell lines. Here, we describe the development and characterization of a macaque cell line that can be infected by all of the strains of SIV that we have tested, including macrophage- and T-cell-tropic strains, primary and cell-line adapted strains, and SIVmac, SIVMne, and SIVsm isolates. The cells can be infected by strains of HIV type 2 (HIV-2) to varying degrees, but not by either cloned or primary isolates of HIV type 1 (HIV-1). This cell line is a derivative of a rhesus macaque mammary tumor cell line (CMMT) engineered to express human CD4. For these studies, a CMMT-CD4 clone expressing an integrated copy of a truncated HIV-1 long terminal repeat fused to the beta-galactosidase gene (LTR-beta-gal) was established to allow detection of infectious SIV after a single round of replication. Here, we demonstrate the ability of the CMMT-CD4-LTR-beta-gal cell line to rapidly and quantitatively detect infectious SIV. Using these cells to assay virus, we could readily measure neutralizing antibody activity in animals infected with different SIV isolates. Neutralizing activity was detected against the homologous virus and lower, but detectable, activity was measured against heterologous virus. Thus, this system, which is highly sensitive and can detect infection by all of the SIV isolates we tested, is a rapid method for detecting infectious virus and quantitating neutralizing antibody activity.

Persistence of simian immunodeficiency virus Mne variants upon transmission.

In macaques infected with a clone of simian immunodeficiency virus (SIV) Mne, viral variants consistently evolve multiple new potential glycosylation sites in the first variable region (V1) prior to the development of AIDS. In the present study, we asked whether viruses with these glycosylation sites persist when they are transmitted to a naive macaque. Variants that evolved after transmission to a recipient macaque were compared with virus that evolved in the donor, which had been infected by cloned SIV Mne. Upon transmission, the specific serine/threonine-rich motifs potentially encoding novel O-linked glycosylation site(s) in V1 were conserved in virus isolated from lymph node, spleen, and liver tissue from the recipient. There was some accumulation of changes in V3 of envelope in virus from the recipient, whereas changes in this region were not observed in virus from the donor macaque. Some variants detected in the tissue of the recipient at necropsy were most closely related to viruses present in the donor inoculum even though these particular variants were not detected early after infection in the recipient's peripheral blood mononuclear cells. Overall, virus with the predominant V1 sequences associated with progression to disease are transmitted to and persist in the recipient animal.

Sequence and complementation analysis of recF genes from Escherichia coli, Salmonella typhimurium, Pseudomonas putida and Bacillus subtilis: evidence for an essential phosphate binding loop.

We have compared the recF genes from Escherichia coli K-12, Salmonella typhimurium, Pseudomonas putida, and Bacillus subtilis at the DNA and amino acid sequence levels. To do this we determined the complete nucleotide sequence of the recF gene from Salmonella typhimurium and we completed the nucleotide sequence of recF gene from Pseudomonas putida begun by Fujita et al. (1). We found that the RecF proteins encoded by these two genes contain respectively 92% and 38% amino acid identity with the E. coli RecF protein. Additionally, we have found that the S. typhimurium and P. putida recF genes will complement an E. coli recF mutant, but the recF gene from Bacillus subtilis [showing about 20% identity with E. coli (2)] will not. Amino acid sequence alignment of the four proteins identified four highly conserved regions. Two of these regions are part of a putative phosphate binding loop. In one region (position 36), we changed the lysine codon (which is essential for ATPase, GTPase and kinase activity in other proteins having this phosphate binding loop) to an arginine codon. We then tested this mutation (recF4101) on a multicopy plasmid for its ability to complement a recF chromosomal mutation and on the E. coli chromosome for its effect on sensitivity to UV irradiation. The strain with recF4101 on its chromosome is as sensitive as a null recF mutant strain. The strain with the plasmid-borne mutant allele is however more UV resistant than the null mutant strain. We conclude that lysine-36 and possibly a phosphate binding loop is essential for full recF activity. Lastly we made two chimeric recF genes by exchanging the amino terminal 48 amino acids of the S. typhimurium and E. coli recF genes. Both chimeras could complement E. coli chromosomal recF mutations.