Protection by humoral elements independent of virus neutralization activity against an influenza virus challenge.

To investigate the protective efficacy of a seasonal trivalent inactivated whole virion influenza vaccine (TIV) adjuvanted with aluminum phosphate (Fluval AB, referred to here as TIV+Al), we immunized mice with the TIV+Al, and as controls, with TIV, TIV+Al combined with Freund adjuvant (TIV+Al+F), inactivated A/PR/8/34(H1N1) (PR8) strain or PBS, and challenged them with a lethal dose of a mouse-adapted PR8 virus. Serum pools from immunized mice were passively transferred to recipient mice that were then challenged similarly. All actively immunized mice survived the challenge. Of recipient mice receiving serum from mice actively immunized with TIV, TIV+Al or TIV+Al+F, 20%, 80%, and 100% survived, respectively. Rates of mortality and morbidity of recipient mice were inversely proportional to the hemagglutination inhibition (HI) antibody level to the vaccine virus in the absence of detectable PR8-specific HI, neuraminidase inhibition (NI) and virus neutralization (VN) antibodies. No cross-reactivity was observed between vaccine and PR8 strains in in vitro HI, NI or VN assays. In splenocytes from TIV+Al-immunized mice production of IFN-γ or granzyme-B protein and mRNA expression increased (p<0.05). Thus, antibodies play a major role in the protection against a mismatched challenge infection independent of HI, NI and VN activity, but cellular immune responses may contribute to full protection in actively immunized mice.

Immunization with a combination of 2 peptides derived from the C5a receptor significantly reduces early atherosclerotic lesion in Ldlr(tm1Her) Apob(tm2Sgy) J mice.

OBJECTIVE: The goal of this study was to assess whether immunization of Ldlr(tm1Her) Apob(tm2Sgy) J mice with 2 peptides located at the N-terminus of the C5a receptor (C5aR), either alone or in combination, is effective in reducing atherosclerotic lesions. METHODS AND RESULTS: Five- to 6-week-old female Ldlr(tm1Her)Apob(tm2Sgy) J mice were immunized using a repetitive immunization multiple sites strategy with keyhole limpet hemocyanin-conjugated peptides derived from the C5aR, either alone (designated as C5aR-P1 [aa 1-21] and C5aR-P2 [aa 19-31]) or in combination (designated as C5aR-P1+C5aR-P2). Mice were fed a high-fat diet for 10 weeks. Lesions were evaluated histologically; local and systemic immune responses were analyzed by immunohistochemistry of aorta samples and cytokine measurements in plasma samples and splenocyte supernatants. Immunization of Ldlr(tm1Her)Apob(tm2Sgy) J mice with these peptides elicited high concentrations of antibodies against each peptide. Immunization with the single peptide inhibited plaque development. Combined inoculation with C5aR-P1+C5aR-P2 had an additive effect on reducing the lesion in the aorta sinus and descending aortas when compared with controls. This effect correlated with cellular infiltration and cytokine/chemokine secretion in the serum or in stimulated spleen cells as well as specific cellular immune responses when compared with controls. CONCLUSIONS: Immunization of mice with C5aR-P1 and C5aR-P2, either alone or in combination, was effective in reducing early atherosclerotic lesion development. The combined peptide is more potential than either epitope alone to reduce atherosclerotic lesion formation through the induction of a specific Treg cell response as well as blockage of monocyte differentiation into macrophages.

Protection of Chinese painted quails (Coturnix chinensis) against a highly pathogenic H5N1 avian influenza virus strain after vaccination.

Chinese painted quails immunized with a single dose (6 µg HA) of inactivated H5N1 (clade 1) influenza vaccine NIBRG-14 and challenged with 100 LD50 of the heterologous A/Swan/Nagybaracska/01/06(H5N1) (clade 2.2) strain were protected, whereas unvaccinated quails died after challenge. No viral antigens or RNA were detected in cloacal swabs from immunized animals. Sera obtained post-immunization gave low titres in serological assays against the vaccine and the challenge viruses. Our results demonstrate the protective efficacy of the NIBRG-14 strain against the challenge virus and the usefulness of these small birds in protection studies of influenza vaccines.

Asymptomatic and Mild SARS-CoV-2 Infections in a Hungarian Outpatient Cohort in the First Year of the COVID-19 Pandemic.

We aimed to estimate the proportion of the population infected with SARS-CoV-2 in the first year of the pandemic. The study population consisted of outpatient adults with mild or no COVID-19 symptoms and was divided into subpopulations with different levels of exposure. Among the subpopulation without known previous COVID-19 contacts, 4143 patients were investigated. Of the subpopulation with known COVID-19 contacts, 594 patients were investigated. IgG- and IgA-seroprevalence and RT-PCR positivity were determined in context with COVID-19 symptoms. Our results suggested no significant age-related differences between participants for IgG positivity but indicated that COVID-19 symptoms occurred most frequently in people aged between 20 and 29 years. Depending on the study population, 23.4-74.0% PCR-positive people (who were symptomless SARS-CoV-2 carriers at the time of the investigation) were identified. It was also observed that 72.7% of the patients remained seronegative for 30 days or more after their first PCR-positive results. This study hoped to contribute to the scientific understanding of the significance of asymptomatic and mild infections in the long persistence of the pandemic.

Adjuvant modulation of the immune response of mice against the LcrE protein of Chlamydophila pneumoniae.

LcrE protein is a TTSS component of Chlamydophila pneumoniae. The immunogenicity and protective effect of recombinant LcrE protein combined either with Freund's or Alum adjuvant were investigated in mice. The immunization with both protocols resulted in a significant reduction of the number of viable C. pneumoniae in the lungs after challenge. Lower IgG2a/IgG1 ratio in Alum-immunized mice suggested a shift towards Th2 type immune response, but the presence of LcrE-specific IFN-gamma-producing cells in LcrE+Alum-immunized mice also indicates Th1 type response. LcrE-specific IgA level was higher in both the sera and the lungs after using Freund's adjuvant. Phenotype of LcrE-specific IFN-gamma-producing cells was CD4+ in Alum- and Freund's-immunized mice, but CD8+ cells were also detected in Freund's-immunized mice. These results confirm that LcrE induces protective immunity in mice. The results also show that Alum is able to activate the CD4+ cell-based cellular immunity, thus it can be regarded as an alternative adjuvant during vaccine screening and a useful adjuvant in a potential protein vaccine against C. pneumoniae infection.

Genotyping of Chlamydia trachomatis from the endocervical specimens of high-risk women in Hungary.

The distribution of different Chlamydia trachomatis serovars in Hungary has not been reported previously. The objective of this study was to determine the distribution and prevalence of C. trachomatis serovars in a high-risk population by genotyping. The endocervical specimens of 484 female sex workers (FSWs) were screened for C. trachomatis by plasmid PCR. Genotyping was performed in all C. trachomatis-positive samples by PCR-based RFLP analysis of the omp1 gene. A total of 32 specimens (6.6 %) were positive for C. trachomatis. Age was an important risk factor for C. trachomatis infection in FSWs. The highest prevalence was detected in women under the age of 20 (18.8 %). All positive specimens were successfully genotyped and seven serovars were identified. The most prevalent was serovar D (34.4 %), followed by E (21.9 %), F (18.8 %), G (9.4 %), J (9.4 %), H (3.1 %) and I (3.1 %). A heterogeneous distribution of C. trachomatis serovars was observed in the study group, where the most common serovars were D, E and F comprising 75 % of the positive samples. This PCR-based RFLP method could be used in epidemiological studies on the prevalence of C. trachomatis infection to provide more information and to compare the serovar distribution among different cohorts.

Expression of bacterial genes and induction of INF-gamma in human myeloid dendritic cells during persistent infection with Chlamydophila pneumoniae.

The interactions between human monocyte-derived dendritic cells (DCs) and Chlamydophila pneumoniae (Cpn) infection were investigated. Cpn infection induced the maturation and functional activation of DCs, and Cpn antigens were present in all of the subpopulations during the maturation process. Chlamydial antigens were detected in DCs during an observation period of 28 days. The exponential production of infectious elementary bodies was not observed. Chlamydial transcripts of the 16S rRNA gene, groEL-1 and omcB genes were expressed, as determined by a quantitative real-time PCR, but the expression of the ftsK gene was limited. DC cultures produced IFN-gamma, but the presence of IFN-gamma in the culture medium was not the major factor that decreased the growth of Cpn, as was shown by neutralization of the IFN-gamma. A cell population identified as producing IFN-gamma had no markers for T, B, natural killer, monocyte cells or macrophages but displayed DC morphology and the expression of specific DC markers, such as CD11c and HLA-DR. These results reveal a persistent infection of DCs with the expression of some, but not cell division-related genes and the production of IFN-gamma that may contribute to the pathomechanism of chronic inflammatory diseases associated with persistent Cpn infection.

Chronic infections and genetic factors in the development of ischemic stroke.

The aim of this study was to examine whether chronic infections and genetic factors of the host play roles in the pathophysiology of acute noncardioembolic ischemic stroke. Blood samples from 59 subjects with ischemic stroke and 52 control patients were investigated by nested PCR for the presence of C. pneumoniae DNA, HCMV DNA and enterovirus RNA, by ELISA for the levels of antibodies to C. pneumoniae, HCMV, HSV, HHV-6, EBV and the inflammatory chemokine IL-8, and by PCR for promoter polymorphism of the IL-8 and CD14 host genes. Associations of stroke with the HCMV IgG and HSV-1 IgA antibody levels were observed. No association of stroke was detected with the presence of C. pneumoniae, HCMV or enterovirus nucleic acids in the peripheral blood, C. pneumoniae IgM, IgG and IgA, the HSV IgG, the EBV IgG, or HHV-6 IgG antibody levels, the pathogen burden, the IL-8 or CD14 promoter polymorphisms, or with the serum levels of IL-8 in the overall study population. These results are consistent with the hypothesis that certain pathogens are involved in the development of ischemic stroke.

Association of Chlamydia pneumoniae with coronary artery disease and its progression is dependent on the modifying effect of mannose-binding lectin.

BACKGROUND: The possible association between coronary artery disease (CAD) and Chlamydia pneumoniae (C pneumoniae) infection is controversial. On the basis of the recent suggestion that mannose-binding lectin (MBL) variant alleles are related to an increased risk of severe atherosclerosis, and on the in vitro interaction of MBL with C pneumoniae, we asked whether MBL might contribute to CAD in conjunction with C pneumoniae. METHODS AND RESULTS: Antibodies to C pneumoniae were measured by immunofluorescence and MBL alleles were determined by polymerase chain reaction technique in samples from 210 patients with CAD and 257 healthy subjects from Hungary collected between 1995 and 1996. A higher percentage of patients with CAD were anti-C pneumoniae positive as compared with the control group (P=0.058). However, at logistic regression analysis adjusted to age, sex, and serum lipid levels, this difference was confined only to subjects carrying MBL variant alleles (P=0.035, odds ratio 2.63, [95% CI: 1.07 to 6.45]). In contrast, no significant difference was seen in those homozygous for the normal MBL allele (P=0.412). During a 65+/-5.8-month follow-up period, major outcomes (new myocardial infarction, and/or bypass operation or cardiovascular death) occurred in 11 C pneumoniae positive and 3 C pneumoniae negative patients. In the C pneumoniae positive group, the odds ratio of development of outcomes was 3.27 (95% CI: 1.10 to 9.71, P=0.033) in the carriers of the MBL variant alleles compared with the homozygous carriers of the normal MBL allele. CONCLUSIONS: These results indicate that infection with C pneumoniae leads mainly to the development and progression of severe CAD in patients with variation in the MBL gene.

High rate of early restenosis after carotid eversion endarterectomy in homozygous carriers of the normal mannose-binding lectin genotype.

BACKGROUND AND PURPOSE: Mannose-binding lectin (MBL) is thought to influence the pathophysiology of cardiovascular disease by decreasing the risk of advanced atherosclerosis and by contributing to enhanced ischemia reperfusion injury. Thus, we investigated the role of MBL in restenosis after eversion endarterectomy in patients with severe carotid atherosclerosis. METHODS: In a prospective study, 123 patients who underwent carotid endarterectomy were followed-up by carotid duplex scan (CDS) sonography for 14 months. In a retrospective study, we examined 17 patients and 29 patients, respectively, who had or had not at least 50% restenosis 29 months after carotid eversion endarterectomy. MBL genotypes were analyzed by a polymerase chain reaction-based method, and MBL serum concentrations were measured. RESULTS: In the prospective study in the patients homozygous for the normal MBL genotype, CDS values were significantly higher after 14 months of follow-up compared with the values measured 6 weeks after surgery (P<0.001). In contrast, only a slight increase was registered in patients carrying MBL variant alleles. The differences were much more pronounced in female than in male patients. Similar differences were observed when patients with high and low MBL serum concentrations were compared. In the retrospective study, a significant increase in the frequency of MBL variant genotypes was observed in patients not experiencing restenosis compared with the patients with restenosis (P=0.007). CONCLUSIONS: These results indicate that reoccurrence of stenosis after carotid endarterectomy is partially genetically determined and imply that MBL contributes significantly to the pathophysiology of this condition.

High-level cellular and humoral immune responses in Guinea pigs immunized intradermally with a heat-inactivated varicella-zoster virus vaccine.

The threat of varicella and herpes zoster in immunocompromised individuals necessitates the development of a safe and effective varicella-zoster virus (VZV) vaccine. The immune responses of guinea pigs to the intradermal (i.d.) or subcutaneous (s.c.) administration of a heat-inactivated or live VZV vaccine were investigated. Relative to nonimmunized animals, a single 399-PFU dose of vaccine induced nonsignificant increases in gamma interferon (IFN-γ), granzyme B, and perforin mRNA expression in the splenocytes of all groups, while two i.d. administrations of the inactivated vaccine increased IFN-γ mRNA expression significantly (P < 0.005). A single 1,995-PFU dose significantly increased the expression of IFN-γ mRNA in the groups receiving the vaccine either i.d. (P < 0.005) or s.c. (P < 0.05), that of granzyme B mRNA in the groups immunized i.d. with the inactivated (P < 0.005) or live (P < 0.005) vaccine, and that of perforin mRNA in the animals that received the inactivated vaccine i.d. (P < 0.005). Importantly, increases in the expression of IFN-γ (P = 0.025), granzyme B (P = 0.004), and perforin (P > 0.05) mRNAs were observed in the animals immunized i.d. with 1,995 PFU of inactivated vaccine relative to those immunized s.c. with the same dose. The proportion of animals expressing IFN-γ mRNA mirrored the proportion expressing IFN-γ protein (correlation coefficient of 0.88). VZV glycoprotein-specific and virus-neutralizing antibodies were produced with no significant intergroup differences. A booster i.d. administration of the 399-PFU dose of heat-inactivated vaccine enhanced the antibody responses. These results demonstrate that i.d. administration of an inactivated VZV vaccine can be an efficient mode of immunization against VZV.

Elevated antibody levels against Chlamydia pneumoniae, human HSP60 and mycobacterial HSP65 are independent risk factors in myocardial infarction and ischaemic heart disease.

The relative significance of traditional risk factors, chronic infections and autoimmune processes in the development of acute myocardial infarction (AMI) has not been fully elucidated. We compared serum IgG antibody titres to various pathogens, i.e. Chlamydia pneumoniae (Cpn), cytomegalovirus (CMV) and herpes simplex virus type 1 (HSV-1), and to the potential autoantigens human heat shock protein 60 (hHSP60) and mycobacterial heat shock protein 65 (mHSP65), in serum samples obtained from patients 3-48 h after AMI (n = 40) or stable effort angina (SEA, n = 43), and from controls (n = 46). The strongest association was observed between AMI and the elevated level of hHSP60 antibodies. The association between AMI and the level of Cpn antibodies was also significant. High levels of hHSP60 and Cpn antibodies represented independent risk factors for the development of AMI, but the simultaneous presence of high levels of antibodies to Cpn and hHSP60 suggested a joint effect on the relative risk of AMI (OR = 12.0-21.1). The antibody titres to mHSP65 were higher in the SEA group than in the controls, and the simultaneous presence of high levels of Cpn and mHSP65 antibodies meant an increased risk among the SEA patients. The antibody titres to CMV or HSV-1 were similar in the three groups. In conclusion, these results demonstrate associations of AMI with high levels of anti-hHSP60 and anti-Cpn antibodies, and of SEA with the level of anti-mHSP65 antibodies, these being independent risk factors.

Chlamydophila (Chlamydia) pneumoniae induces histidine decarboxylase production in the mouse lung.

Chlamydophila (Chlamydia) pneumoniae (C. pneumoniae) is the third most common cause of community-acquired pneumonia and is probably involved in the development of certain chronic inflammatory diseases, including atherosclerosis and adult-onset asthma. Histamine, synthesized by histidine decarboxylase (HDC) from L-histidine, plays an essential role in allergic and inflammatory processes and in cell differentiation. The effect of C. pneumoniae infection on the expression of HDC has not been examined. In the present study, normal Balb/c mice and HDC knockouts, and control mice with a CD1 background were infected intranasally with C. pneumoniae. On days 1, 3, 7, 16 and 31 after infection, the normal Balb/c mice were sacrificed and divided into three groups. In the homogenized lungs of the first group, C. pneumoniae titres were determined and demonstrated peak levels on day 7. HDC production was revealed by a Western blot assay throughout the observation period of 1-16 days, and cytokine concentrations were determined by ELISA. The interleukin-3 (IL-3) and interleukin-6 (IL-6) levels were highest on day 1 and on days 1-3, respectively; the interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) levels reached the maximum on day 7, but the quantity of IL-4 was still three times higher than that in the control group 16 days after infection. The lungs of the mice in the second group were processed for the in situ demonstration of HDC activity, while the lungs in the third group were stained for C. pneumoniae antigen. The HDC activity was increased predominantly in the bronchial epithelial cells, while C. pneumoniae antigens were expressed especially in the interstitial macrophages. The HDC knockout mice exhibited a higher survival rate after C. pneumoniae infection than did the control mice. These results point to a strong association between local histamine production and other inflammatory mediators and are novel in demonstrating the role of histamine in the pathomechanism of C. pneumoniae infections.

Fast vaccine design and development based on correlates of protection (COPs).

New and reemerging infectious diseases call for innovative and efficient control strategies of which fast vaccine design and development represent an important element. In emergency situations, when time is limited, identification and use of correlates of protection (COPs) may play a key role as a strategic tool for accelerated vaccine design, testing, and licensure. We propose that general rules for COP-based vaccine design can be extracted from the existing knowledge of protective immune responses against a large spectrum of relevant viral and bacterial pathogens. Herein, we focus on the applicability of this approach by reviewing the established and up-coming COPs for influenza in the context of traditional and a wide array of new vaccine concepts. The lessons learnt from this field may be applied more generally to COP-based accelerated vaccine design for emerging infections.

Immunization of Chlamydia pneumoniae (Cpn)-infected Apob(tm2Sgy)Ldlr(tm1Her)/J mice with a combined peptide of Cpn significantly reduces atherosclerotic lesion development.

OBJECTIVE: To investigate the antigenic effect of a peptide containing two epitopes of Chlamydia pneumoniae (Cpn) on atherosclerotic lesion formation in mice infected with Cpn. MATERIALS AND METHODS: Six-week-old Apob(tm2Sgy)Ldlr(tm1Her)/J mice were immunized using a repetitive immunization multiple-sites strategy with KLH-conjugated peptides derived from the major outer membrane protein and the putative outer membrane protein 5 of Cpn. Mice were fed a high-fat diet and infected with Cpn twice during the 10-week diet period. Lesions were evaluated histologically; local and systemic immune responses were analyzed by immunohistochemistry of aorta samples and cytokine measurements in plasma samples and splenocyte supernatants. RESULTS: Mice immunized with the combined Cpn peptide showed a greater reduction in lesion size compared to mice immunized with either epitope alone [54.7% vs 39.8% or 41.72%] and was also associated with a significant decrease in lesion area in descending aortas compared with those in controls (88.9% for combined Cpn peptide, 81.9% for MOMP peptide and 75.7% for Omp5, respectively). This effect was associated with a shift in the cellular composition of plaques towards decreased inflammatory cell and increased regulatory T-cell content. Additionally, the effect was also connected with decreased secretion of proinflammatory cytokines and increased production of anti-inflammatory cytokines demonstrated in plasma and in supernatant on stimulated spleen cells. CONCLUSIONS: Atherosclerotic lesion formation may be promoted by Cpn infection in the presence of a high-fat diet, and reduced by immunization with the combined Cpn peptide. The combined peptide has more potential than either epitope alone in reducing atherosclerotic lesion development through Treg expansion.

Human cytomegalovirus neutralising antibodies and increased risk of coronary artery disease in Indian population.

BACKGROUND: Several studies have reported a conflicting association between cytomegalovirus (CMV) infection and coronary artery disease (CAD) based on the levels of total anti-CMV antibodies. However, none have estimated the levels of specific neutralising antibodies (NA) to CMV, which may be clinically more relevant. OBJECTIVE: To determine whether CMV-NA titres show a better association with CAD compared with total anti-CMV antibody levels. DESIGN: CMV-NA titres were measured by micro-neutralisation assay and anti-CMV IgG antibodies using ELISA in 391 consecutive CAD patients compared with the same number of controls (N=782), and 91 patients reporting recurrent cardiac events during a 4-year follow-up compared with those without a recurrent event (N=182). Levels of inflammatory markers, interleukin 6, high-sensitivity C reactive protein, fibrinogen and secretory phospholipase A2 (sPLA2), were measured by ELISA. Analysis of variance and logistic regression were used for statistical analyses. RESULTS: High CMV-NA titres showed a positive association with CAD occurrence (OR 2.24, 95% CI 1.31 to 3.85, p=0.003) and recurrent cardiac events in CAD patients (OR 4.65, 95% CI 1.21 to 17.86, p=0.025) compared with total CMV antibodies (OR 1.67, 95% CI 1.04 to 2.69, p=0.034, and 2.70, 1.04 to 7.02, p=0.040, respectively). Patients with higher quartile of CMV-NA titres and sPLA2 levels had an adjusted OR of 7.82 (95% CI 1.87 to 32.65, 0.005) for recurrent cardiac events compared with those with the lowest quartiles for both markers. CONCLUSION: These findings suggest that high CMV-NA titres in combination with inflammatory markers improve prediction of cardiac events in the Asian Indian population.

Impact of multiple antigenic epitopes from ApoB100, hHSP60 and Chlamydophila pneumoniae on atherosclerotic lesion development in Apob(tm2Sgy)Ldlr(tm1Her)J mice.

AIMS: To assess whether immunizing Apob(tm2Sgy)Ldlr(tm1Her)J mice simultaneously with different atherosclerosis-related epitopes engineered in a single recombinant protein is effective in reducing atherosclerotic lesions. METHODS AND RESULTS: Antigenic epitopes were incorporated into a dendroaspin scaffold: AHC (ApoB100 peptide + hHSP60 peptide [hHSP60(153-163)] + putative epitope derived from Chlamydophila pneumoniae [Cpn]) and AHHC (AHC + hHSP60(303-312)); and were compared with construct A (ApoB peptide), construct H (hHSP60(153-163)), and construct AH (ApoB100 peptide + hHSP60(153-163)). Immunization with 2 multiple-antigenic epitope constructs elicited high levels of antibodies against each epitope in Apob(tm2Sgy)Ldlr(tm1Her)J mice (apart from hHSP60(153-163), which induced a low antibody response). Histological analyses demonstrated that the mice immunized with AHHC and AHC showed significant reductions in the size of atherosclerostic lesions compared with controls (63.8% and 63.2%; P < 0.001, respectively), and significantly greater reductions in lesions size compared with those after immunization with construct A (24.9%; P < 0.01), H (26.8%; P < 0.05), and AH (42.9%; P < 0.001). Moreover, combination of 2 short Cpn peptides along with ApoB and hHSP60 peptides had an additive effect on reducing the lesion without Cpn infection. Reduction in plaque size correlated with cellular infiltration and cytokine/chemokine secretion in serum or by stimulated spleen cells as well as specific cellular immune responses when compared with controls. CONCLUSIONS: Immunization of mice with a single construct containing multiple epitopes derived from ApoB100, hHSP60 and Cpn was more effective in reducing early atherosclerotic lesions through the induction of a specific Treg-cell response than was the construct containing either mono- or bi-epitopes. This approach offers attractive opportunities for the design of protein-based, multivalent vaccines against atherosclerosis.

Immunization with a combination of ApoB and HSP60 epitopes significantly reduces early atherosclerotic lesion in Apobtm2SgyLdlrtm1Her/J mice.

OBJECTIVE: HSP60 is emerging as an immunodominant target of autoantibodies in atherosclerosis and recent studies have revealed oxLDL as a key antigen in the development of atherosclerosis. In this study, we assay whether immunizing Apobtm2SgyLdlrtm1Her/J mice with a combination of ApoB and human HSP60 peptides has an additive effect on atheroprotection compared to ApoB or HSP60 peptides applied alone by following atherosclerotic lesion development. METHODS AND RESULTS: In this study, 2 weeks after the first immunization, Apobtm2SgyLdlrtm1Her/J mice were placed on a high-fat diet for 8 weeks followed by 2 weeks on a normal diet allowing the mice to adapt to the environment before sacrifice. High levels of ApoB and HSP60 antibodies were detectable in week 2 and week 12 following the first immunization with KLH-conjugated ApoB and HSP60 peptides either individually or in combination. Histological analyses demonstrated that mice immunized with both, ApoB and HSP60 peptides, showed the most significant reduction in atherosclerotic lesions (41.3%; p<0.001) compared to a reduction of 14.7% (p<0.05) and 21.1% (p<0.01) in mice immunized with ApoB or HSP60 peptides, respectively; control mice were immunized with either PBS or adjuvant alone. These results were further supported by significant differences in the cellular and humoral immune responses between test animals. CONCLUSIONS: Immunization with a combination of ApoB and HSP60 peptide antigens significantly reduced early atherosclerotic lesions in the Apobtm2SgyLdlrtm1Her/J mouse model of atherosclerosis. This approach offers promise as a novel strategy for developing anti-atherosclerotic agents.

Chlamydophila pneumoniae and human cytomegalovirus in atherosclerotic carotid plaques--combined presence and possible interactions.

The aim of our study was to investigate the combination of Chlamydophila pneumoniae and human cytomegalovirus (HCMV) as a pathogenic factor in atherosclerosis. Accordingly, we tested by means of PCR and immunohistochemistry the presence of these pathogens in the same atherosclerotic carotid specimen. The histology of the samples and the patients' antibodies against these pathogens were evaluated. Further, we examined the impact of C. pneumoniae and HCMV infection on the gene expression of the human monocytic cell line U937. Six of the 22 samples contained only C. pneumoniae, 4 contained only HCMV, 7 contained both C. pneumoniae DNA and/or antigens of both pathogens, and 5 samples were negative. No correlation was found between the presence of these microbes and either the cellular structure of the plaques, or the serostatus of the patients. The infection of U937 cells with HCMV and especially C. pneumoniae induced inflammation and atherosclerosis-related genes. Furthermore, the doubly-infected cells produced higher levels of the mRNA of pro-platelet basic protein and fatty acid binding protein 4. In conclusion, C. pneumoniae is often present in combination with HCMV in atherosclerotic carotid lesions. The in vitro coinfection model reveals that the doubly-infected monocytes are potent expressors of proatherosclerotic genes, suggesting that this coinfected population may accelerate the process of atherosclerosis.