Effects of estradiol and progesterone on susceptibility and early immune responses to Chlamydia trachomatis infection in the female reproductive tract.

We have used a previously described rodent model to examine the influence of hormonal environment on susceptibility and immune responses to genital Chlamydia infection. Ovariectomized rats were administered estradiol, progesterone, or a combination of both, infected with Chlamydia trachomatis via the intrauterine route, and sacrificed 5 days later. Histopathological examination showed severe inflammation in the uteri and vaginae of progesterone-treated animals, whereas animals receiving estradiol or a combination of both hormones showed no inflammation. Large numbers of chlamydiae were found in vaginal secretions of progesterone-treated and combination-treated animals, while estradiol-treated animals had none. Tissue localization showed that numerous chlamydial inclusions were present in the uterine epithelium of the progesterone group and the cervicovaginal epithelium of the combination group. Examination of the acute immune responses of the infected animals showed that maximum activation was present in the draining lymph node cells from the progesterone-treated group, and these cells were producing large amounts of interleukin-10 and gamma interferon compared to other hormone-treated groups. In contrast, spleen cell proliferation was suppressed in progesterone-treated animals compared to other hormone-treated groups. We conclude that progesterone increases and estradiol decreases susceptibility to intrauterine chlamydial infection in this rat model. Our data demonstrate that hormone environment, at the time of infection, has a profound effect on the outcome of microbial infection in the female reproductive tract.

Use of a mouse lung challenge model to identify antigens protective against Chlamydia pneumoniae lung infection.

Chlamydia pneumoniae is emerging as a significant human pathogen. Infection causes a range of respiratory tract diseases and is associated with atherosclerosis. A vaccine could provide a considerable public health benefit; however, antigens able to elicit a protective immune response are largely unknown. A panel of open-reading frames (ORFs) from the C. pneumoniae genome sequence was screened for ability to elicit protective responses. Balb/c mice immunized with DNA containing the ORFs were tested for their ability to limit lung infection following an intranasal challenge. Immunization with DNA encoding the major outer membrane protein or an ADP/ATP translocase (Npt1(Cp)) of C. pneumoniae resulted in a reduced bacteria load in the lung after challenge. The identification of these antigens as protective is a significant step toward development of a C. pneumoniae vaccine and demonstrates the feasibility of using a DNA immunization strategy to screen the C. pneumoniae genome for other protective ORFs.

Chlamydia pneumoniae infection significantly exacerbates aortic atherosclerosis in an LDLR-/- mouse model within six months.

We have previously shown that infection with the C. pneumoniae AR39 strain once monthly for 9 consecutive months significantly exacerbated atherosclerosis in mice with LDL receptor deficiency (LDLR-/-) in the presence of a high cholesterol diet. To further optimize the LDLR-/- mouse model for studying the mechanisms of C. pneumoniae atherogenesis, we have tested a different infection protocol with intranasal inoculation twice monthly for 6 consecutive months in the present study. We found that C. pneumoniae infection for 6 months was sufficient to produce a 130%, significantly greater exacerbation of aortic atherosclerosis in LDLR-/- mice in the presence of a high cholesterol diet. Mice receiving a high cholesterol diet alone displayed a lesion area index of 18.2 +/- 6.1 (S.D.) while mice treated with both the high cholesterol diet and C. pneumoniae infection had a lesion area index of 41.8 +/- 15.2 (S.D.). However, the chlamydial infection did not significantly alter the mouse serum total cholesterol or the LDL levels induced by the high cholesterol diet. This study not only confirms our previous findings that C. pneumoniae infection can exacerbate aortic atherosclerosis lesion in the LDLR-/- mice, but also further optimizes the LDLR-/- mouse model for future mechanism studies.

Chlamydia trachomatis infection in the female reproductive tract of the rat: influence of progesterone on infectivity and immune response.

As the most common cause of sexually transmitted disease in women, chlamydial infections can lead to pelvic inflammatory disease, infertility, and ectopic pregnancy. To better understand the role played by sex hormones in modulating the immune response of the genital tract to microbial infections, we have developed a rat model to study Chlamydia trachomatis infection. Inbred female Lewis rats were primed with progesterone and inoculated by intrauterine instillation of C. trachomatis (mouse pneumonitis strain MoPn) into each uterine horn. When infected animals were examined for the presence of chlamydial antigens 14 days postinfection, both the uterus and vagina were found to be positive compared to those of saline-treated animals, which did not show specific staining. The involvement of local and systemic immune systems following chlamydial infection was determined by analyzing major histocompatibility complex (MHC) class II expression in the reproductive tract and lymphocyte proliferation in response to mitogenic and chlamydia-specific stimulation of cells from the spleen and lymph nodes (LN) draining the reproductive tract. Enhanced proliferation was observed in LN following mitogenic but not antigenic (MOMP [major outer membrane protein]) stimulation. In contrast, spleen cell proliferation was lower in chlamydia-infected rats than in saline-treated controls. MHC class II expression, an indicator of inflammatory responses, was upregulated in the uterus, on glandular epithelial cells, and adjacent to glands in response to chlamydial infection. In other experiments, when rats were infected at estrus and diestrus without prior progesterone priming, chlamydial inclusions were not detected in either the uterus or vagina. However, enhanced lymphocyte proliferation was observed in response to mitogenic and MOMP stimulation in the reproductive tract-draining LN from estrous and diestrous animals. These findings indicate that under appropriate endocrine conditions, the rat uterus is susceptible to C. trachomatis infection and that immune responses to this pathogen can be detected locally and systemically. Further, they suggest that clearance of the infection from the reproductive tract involves immune cells from the LN draining the reproductive tract.

Inactivated poliovirus vaccine: past and present experience.

Many countries have made use of inactivated poliovirus vaccine (IPV) in their national poliovirus control programs since 1955. Until 1961 IPV was the only vaccine available for the control of poliovirus, but subsequently many countries opted to use the Sabin attenuated poliovirus vaccine (OPV), which was perceived as more effective in preventing intestinal infection and in ensuring community protection by spreading to unvaccinated contacts of vaccinees. Nevertheless, IPV has remained the vaccine of choice in several countries, where experience has shown that it represents a safe and effective option for disease control. IPV limits subsequent infection of the pharynx and intestine in vaccinees, and is able to control circulation of poliovirus in a vaccinated population, providing effective community protection. Furthermore IPV contains only killed virus and cannot cause vaccine-associated paralytic poliomyelitis as OPV sometimes does. This paper reviews the history of the use of IPV, with emphasis on its efficacy and its ability to safely protect communities in which it is used. As the incidence of poliomyelitis declines new control strategies should take account of the knowledge of the use of poliovirus vaccines acquired since 1955.

Poliovirus hybrids expressing neutralization epitopes from variable domains I and IV of the major outer membrane protein of Chlamydia trachomatis elicit broadly cross-reactive C. trachomatis-neutralizing antibodies.

Trachoma and sexually transmitted diseases caused by Chlamydia trachomatis are major health problems worldwide. Epitopes from the variable domains of the major outer membrane protein are candidates for vaccine development. We have constructed hybrid polioviruses expressing sequences from major outer membrane protein variable domains I and IV. Antisera to the hybrids could, in combination, strongly neutralize 8 of the 12 C. trachomatis serovars most commonly associated with oculogenital infections and weakly neutralize the others.

Mouse neurovirulence determinants of poliovirus type 1 strain LS-a map to the coding regions of capsid protein VP1 and proteinase 2Apro.

Poliovirus type 1 strain LS-a [PV1(LS-a)] is a OV variant adapted to mice by multiple passages through mouse and monkey tissues. To investigate the molecular basis underlying mouse neurovirulence of PV1(LS-a), a cDNA of the viral genome containing nucleotides 112 to 7441 was cloned, and the nucleotide sequence was determined. Compared with that of the mouse avirulent progenitor PV1(Mahoney), 54 nucleotide changes were found in the genome of the PV1(LS-a) virus, resulting in 20 amino acid substitutions in the virus polyprotein. Whereas the nucleotide changes were scattered throughout the genome, the amino acid substitutions were largely clustered in the capsid proteins and, to a certain extent, in the virus proteinase 2Apro. By in vitro mutagenesis, PV1(LS-a)-specific capsid mutations were introduced into a cDNA clone of PV1(Mahoney). We show that neither the individual amino acid mutations nor combinations of mutations in the region encoding VP1 conferred to PV1(Mahoney) the mouse-adapted phenotype of PV1(LS-a). Chimeric cDNA studies demonstrated that a recombinant type 1 virus containing the PV1(LS-a) sequence from nucleotide 2470 to nucleotide 3625 displayed a neurovirulent phenotype in mice. Further dissection of this region revealed that mouse neurovirulence of PV1(LS-a) was determined by multiple mutations in regions encoding both viral proteinase 2Apro and capsid protein VP1. The mouse neurovirulent viruses, PV1(LS-a), W1-M/LS-Pf [nucleotides 496 to 3625 from PV1(LS-a)], and W1-M/LS-NP [nucleotides 2470 to 3625 from PV1(LS-a)], showed increased sensitivity to heat treatment at 45 degrees C for 1 h. Surprisingly, the thermolabile phenotype was also displayed by a recombinant of PV1(Mahoney) carrying a PV1(LS-a) DNA fragment encoding the N-terminal portion of 2Apro. This suggests that base substitutions in the region encoding 2Apro affected capsid stability, thereby contributing to the neurovirulence of the virus in mice.

A poliovirus hybrid expressing a neutralization epitope from the major outer membrane protein of Chlamydia trachomatis is highly immunogenic.

Trachoma and sexually transmitted diseases caused by Chlamydia trachomatis are major health problems worldwide. Epitopes on the major outer membrane protein (MOMP) of C. trachomatis have been identified as important targets for the development of vaccines. In order to examine the immunogenicity of a recombinant vector expressing a chlamydial epitope, a poliovirus hybrid was constructed in which part of neutralization antigenic site I of poliovirus type 1 Mahoney (PV1-M) was replaced by a sequence from variable domain I of the MOMP of C. trachomatis serovar A. The chlamydial sequence included the neutralization epitope VAGLEK. This hybrid was viable, grew very well compared with PV1-M, and expressed both poliovirus and chlamydial antigenic determinants. When inoculated into rabbits, this hybrid was highly immunogenic, inducing a strong response against both PV1-M and C. trachomatis serovar A. Antichlamydia titers were 10- to 100-fold higher than the titers induced by equimolar amounts of either purified MOMP or a synthetic peptide expressing the VAGLEK epitope. Furthermore, rabbit antisera raised against this hybrid neutralized chlamydial infectivity both in vitro, for hamster kidney cells, and passively in vivo, for conjunctival epithelia of cynomolgus monkeys. Because poliovirus infection induces a strong mucosal immune response in primates and humans, these results indicate that poliovirus-chlamydia hybrids could become powerful tools for the study of mucosal immunity to chlamydial infection and for the development of recombinant chlamydial vaccines.

Poliovirus antigenic hybrids simultaneously expressing antigenic determinants from all three serotypes.

We have constructed six hybrid polioviruses (PVs) modified to express PV type 2 and type 3 antigenic determinants on a PV type 1 (Mahoney) capsid. The hybrids were modified in neutralizing antigenic site (NAg) I and/or NAgII. They were viable, but impaired for growth in comparison to PV1 (Mahoney). Some hybrids modified to express type 2 and type 3 NAgI determinants simultaneously displayed some type 2 but no type 3 antigenicity (in addition to type 1 antigenicity associated with other antigenic sites). Hybrids modified to express a type 2 NAgI determinant and a type 3 NAgII determinant, or vice versa, displayed antigenic characteristics of all three serotypes, although expression of the modified NAgII determinant was weak. We conclude that it is possible to construct a viable hybrid PV simultaneously modified in NAgI and NAgII which expresses antigenic determinants of all three serotypes.

Construction and characterization of a poliovirus/rhinovirus antigenic hybrid.

In order to study the properties of foreign antigenic sites expressed on poliovirus a hybrid was constructed in which neutralization antigenic site IA of poliovirus type 1 strain Mahoney [PV1(M)] was replaced by neutralization immunogenic site IA (NImIA) of human rhinovirus 14 (HRV14). The resulting hybrid was viable, but growth was impaired in comparison to PV1(M). The hybrid expressed both PV1(M) and HRV14 antigenic determinants. When inoculated into rabbits it elicited neutralizing antibodies against both PV1(M) and HRV14. Furthermore, the hybrid was efficiently neutralized by polyclonal antisera specific for either PV1(M) or HRV14 and by three out of five monoclonal antibodies directed to NImIA. The monoclonal antibodies also blocked binding of the hybrid to the cellular receptor for poliovirus. One of them is thought to neutralize rhinovirus in this manner, and it appears that NImIA is expressed in a sufficiently favorable context on the hybrid for the same mechanism to be effective. This can be interpreted to mean that the interactions between the parental viruses and their respective cellular receptors are very similar.

The effect of site and mode of expression of a heterologous antigenic determinant on the properties of poliovirus hybrids.

The antigenic site normally situated in the EF loop of VP2 (2EF) of poliovirus type 2 (Lansing) [PV2 (L)] was expressed in 2EF or in the BC loop of VP1 (1BC) of PV1 (Mahoney) [PV1 (M)]. A hybrid virus expressing the site in 2EF of PV1 (M) is known to be neutralizable by PV2 (L)-specific antisera and to induce neutralizing antibodies against PV-2 (L). In contrast, a hybrid expressing a related sequence in 1BC of PV1 (M), which maintained the length of the native BC loop, was not neutralizable by PV2 (L)-specific antisera and did not induce PV2 (L)-neutralizing antibodies. However, when 1BC was extended, so that it was longer than the native 1BC, the resulting hybrids induced low titers of PV2 (L)-neutralizing antibody although they were still not neutralizable by PV2 (L)-specific antisera. Synthetic peptides copying the extended sequences raised neutralizing antibodies which were able to distinguish between the sequence expressed in the extended 1BC, in the native-length 1BC and in 2EF. The growth rates of the hybrids with modifications to 1BC depended upon the nature of the modifications. The hybrid with the native length 1BC grew poorly, but extending 1BC tended to improve the growth rate, and extending 1BC with PV1 (M)-specific sequences nearly restored wild-type growth rates. Thus, the size, precise sequence and location of a heterologous antigenic site expressed on poliovirus have significant effects on the properties of that determinant and of the hybrid expressing it. Antigenicity of hybrids can be modified and their growth rate can be enhanced by appropriate choices of these parameters.

Phenotypic characterization of antigenic hybrids of poliovirus.

Three poliovirus hybrids, modified in neutralization antigenic sites (NAgs) I or II, were characterized for several phenotypic traits. The modifications to the capsid interfered with some stage of the life-cycle of the virus, since all three hybrids were growth-impaired in comparison to poliovirus type 1 (Mahoney) [PV1 (M)], the wild-type parent virus. All hybrids exhibited a reduced growth rate and a small-plaque phenotype, but they were not temperature sensitive. Furthermore, only one hybrid was slightly less stable to heating than the parent virus; the other two were as stable as the parent. Therefore, decreased thermal stability of the capsid is not an important cause of the poor growth characteristics of these hybrids.

Construction of a poliovirus type 1/type 2 antigenic hybrid by manipulation of neutralization antigenic site II.

There are three serotypes of poliovirus, poliovirus type 1 (PV-1), PV-2, and PV-3. These viruses each display four distinct neutralization antigenic sites, designated N-AgI, N-AgII, N-AgIIIA, and N-AgIIIB. It has been demonstrated previously that part of N-AgI can be replaced with heterogeneous amino acid sequences, resulting in hybrid viruses expressing heterogeneous antigenic determinants. To study whether hybrid viruses could be constructed by modifying another antigenic site, a part of N-AgII (amino acids 158 to 173 of VP2) of PV-1(Mahoney) was replaced with the equivalent sequence from PV-2(Lansing). The resulting hybrid was viable and expressed both PV-1 and PV-2 antigenic determinants. When inoculated into rabbits, the hybrid induced neutralizing antibodies against both PV-1 and PV-2, showing that amino acids 158 to 173 of VP2 are able to function as an antigenic site independent of the rest of N-AgII. Manipulation of N-AgII represents a useful alternative method for the production of hybrid polioviruses.

Synthetic peptide vaccines against foot-and-mouth disease. I. Duration of the immune response and priming in guinea-pigs, rabbits and mice.

The immunogenicity of two aphthovirus-specific synthetic peptides was investigated. One peptide copied the sequence of amino acids 141 to 160 from the capsid VP1 of the aphthovirus strains O1 BFS 1860 and O1 Kaufbeuren (O peptide), the other copied the equivalent sequence from aphthovirus strain A24 Cruzeiro (A peptide). Peptide coupled to keyhole limpet haemocyanin (KLH) stimulated a long-lasting immune response in guinea-pigs and rabbits. Significant levels of antibody were detectable at least one year after vaccination, although the reactivity of the antibody depended on the species and the peptide used. In some circumstances the peptides were able to prime the immune system such that a subsequent dose of peptide boosted antibody production. This effect, also, was dependent on the species of experimental animal and on the peptide used, an observation which has important implications for the use of such peptides as vaccines.

Synthetic peptide vaccines against foot-and-mouth disease. II. Comparison of the response of guinea-pigs, rabbits and mice to various formulations.

The immune response of guinea-pigs to vaccination with either of two aphthovirus-specific synthetic peptides was investigated. One peptide copied the sequence of amino acids 141 to 160 from the VP1 of aphthovirus strains O1BFS 1860 and O1 Kaufbeuren (O peptide), the other copied the equivalent sequence from aphthovirus strain A24 Cruzeiro (A peptide). The immune response was enhanced when the peptides were conjugated to a carried protein, but the choice of carrier protein and cross-linking agent was not critical in obtaining enhancement. The response was greatest when the peptide or peptide-carrier conjugate was adjuvanted in Freund's complete adjuvant (FCA). The O peptide was poorly immunogenic and did not confer protection against challenge with infectious virus, whereas the A peptide had good immunogenicity and did confer protection. This reflected the relative immunogenicity of the parent viruses. Rabbits, three strains of guinea-pigs and seven strains of mice were vaccinated with the O peptide conjugated to keyhole limpet haemocyanin (KLH). Considerable variation was observed between the responses of each strain and it is proposed that this relates to their repertoire of immune response genes.

Synthetic peptide vaccines against foot and mouth disease.

Synthetic peptide vaccines against foot-and-mouth disease (FMD) have been under examination for several years. Recent reports indicate that effective synthetic vaccines for use in cattle are being developed. However, there are still some unresolved questions regarding the response of cattle and other animals to these vaccines. This brief review describes some recent studies of synthetic vaccines against FMD and draws attention to some deficiencies in our understanding of the response to these vaccines.

Isolation of capsid proteins of foot-and-mouth disease virus by chromatofocusing.

A method for the isolation of foot-and-mouth disease virus (FMDV) capsid proteins was developed. The FMDV capsid proteins VP1, VP2, VP3 and VP0 were isolated from sucrose gradient purified virus by chromatofocusing in a pH 7.4-4.0 gradient on Polybuffer exchanger PBE 94. Under the conditions used the proteins eluted in the sequence VP1, VP2, VP0 (when present) and VP3. Capsid protein VP4 did not elute and could not be isolated by this method. Protein concentration in the eluate was monitored by the use of a radiolabelled marker and recoveries of approximately 50% of the input marker could be achieved when using up to 15 mg of virus and a 30-ml column. The high capacity and relative simplicity of chromatofocusing make it a useful alternative to other methods of purifying proteins.