Attenuated Mycoplasma bovis strains provide protection against virulent infection in calves

Mycoplasma bovis is a major etiological agent of pneumonia and arthritis in feedlot beef cattle. To develop a novel live vaccine against M. bovis, two attenuated M. bovis strains, P150 and P180, were tested in calves for protection against challenge with the virulent M. bovis parental strain. Twenty calves were divided into four groups of five calves each that were designated as the P150, P180, positive control (PC), and negative control (NC) groups. Each calf in the P150 and P180 groups was immunized with 10⁹ CFU of P150 or P180, respectively, via the nasal cavity, and the PC and NC groups received the mock inoculation. Baseline data were collected for 46 days post-immunization. The clinical signs were scored, and rectal temperatures and daily weight gain were recorded. The blood leukocyte count, the neutrophil ratio, and the serum levels of IgG, IgA, IFN-β, and TNF-α were quantified using laboratory tests, and the nasal shedding was evaluated using microbiological methods. The P150, P180, and PC calves were challenged with a dose of 10¹⁰ CFU of virulent M. bovis by intratracheal injection on 3 consecutive days. The calves were monitored for 25 days post-challenge to observe changes in the baseline parameters. On day 25 post-challenge the calves were euthanized for necropsy and analysis of tissue samples. The P150 and P180 immunizations caused no clinical abnormality, and did not affect daily weight gain. The post-inoculation neutrophil ratio and serum levels of IgG and IFN-β significantly increased in the P150, P180, and PC calves, whereas the serum levels of IgA and TNF-α did not. After challenge, the PC group developed the typical clinical signs and pathology associated with M. Bovis infection, whereas immunization with P150 or P180 provided efficacious protection. Based on the scores for gross pathology and lung pathology, the protection rates of the P150 and P180 immunizations were 87.7% and 70.8%, respectively. The P150 attenuated strain is a promising candidate for a live vaccine against M. bovis infection in cattle.     

Vaccination of feedlot cattle with extracts and membrane fractions from two Mycoplasma bovis isolates results in strong humoral immune responses but does not protect against an experimental challenge

Mycoplasma bovis is one of the most significant contributors to the bovine respiratory syndrome (BRD) that causes major losses in feedlot and dairy farms. Current experimental vaccines against M. bovis are ineffective and in some cases seem to enhance disease. Experimental infection with M. bovis induces a predominantly Th2 response and high levels of IgG1, which is an inferior opsonin and hence lacks protective capacity. In an attempt to induce a balanced (Th1/Th2) immune response, we have used CpG ODN 2007 as an adjuvant in a trial involving vaccination of cattle with M. bovis total extracts and/or membrane fractions and subsequent intranasal inoculation with an infective dose of M. bovis prepared from two different clinical isolates. Significant IgG1 serum responses were observed against both, extracts and fractions while IgG2 responses were significant against the extracts only. Proliferation of peripheral blood mononuclear cells (PBMC) after incubation with M. bovis cells was only observed in post-challenge samples of cattle vaccinated with both extracts and fractions but not in samples of cattle immunized with the membrane fractions alone. All groups showed transient weight losses and increased temperatures however, there were no significant differences in clinical parameters and survival rates between the groups.     

Blinded, controlled field trial of two commercially available Mycoplasma bovis bacterin vaccines in veal calves

Mycoplasma bovis is an etiologic agent of pneumonia, arthritis, and otitis in young calves, such as those found in the special-fed veal industry. We conducted a blinded, controlled trial of two commercially available M. bovis bacterin vaccines for the prevention of respiratory disease in calves associated with M. bovis infection. Calves were randomly assigned to a subcutaneous treatment of vaccine A (n = 50), adjuvant A (n = 50), vaccine B (n = 50), or 0.9% sterile saline solution (n = 50) beginning at 27 days of age. Upper-respiratory tract colonization was not impacted by vaccination status. Vaccine A significantly reduced the presence of lung lesions (p = 0.0325), however there was no significant reduction of M. bovis in lung lesions. Vaccine B did not significantly reduce total lung lesions or M. bovis-specific lung lesions. The relative risk was determined to be 0.56, 1.0, and 1.36 for vaccine A, adjuvant A, and vaccine B, respectively. There was no association between the total specific antibody isotype (IgM, IgG1, IgG2, IgA) concentrations or M. bovis antibodies and the M. bovis-associated morbidity in the veal calves. Under the field conditions of this study, observed vaccine efficacy for vaccine A and vaccine B was 44% and less than 1%, respectively.     

Ascaris suum infection negatively affects the response to a Mycoplasma hyopneumoniae vaccination and subsequent challenge infection in pigs

Since their first introduction more than a century ago, vaccines have become one of the most cost-effective tools to prevent and manage infectious diseases in human and animal populations. It is vital to understand the possible mechanisms that may impair optimal vaccine efficacy. The hypothesis posed in this study was that a concurrent Ascaris suum infection of pigs vaccinated with a Mycoplasma hyopneumoniae (Mh) vaccine would modulate the protective immune response to a subsequent challenge infection. Four groups of pigs were either (1) untreated (group C), (2) vaccinated against Mh 3 weeks after the start of the study (group V), (3) given a trickle infection with A. suum throughout the study (group A), or (4) given a trickle infection with A. suum and vaccinated against Mh (group AV). All pigs were subsequently inoculated with live Mh bacteria 4 weeks after the Mh vaccination and necropsied after another 4 weeks. All pigs in group V sero-converted 3 weeks after vaccination (100%), as opposed to only 33% of group AV pigs that were Mh-vaccinated and given A. suum. At the end of the study, only 78% of pigs in group AV had sero-converted. Pigs in group AV had a higher mean percentage of lung pathology and the variation was significantly higher in these pigs compared to pigs in group V. The pattern of gene expression in the lungs and draining lymph nodes indicated a local Th2-skewed response induced by A. suum. Our study indicated that A. suum significantly compromised the effect of Mh vaccination. The impact of reduced vaccine efficacy caused by a common gastrointestinal helminth emphasises the importance of parasite control. More focus should be put into this area of research to outline the practical consequences of this interaction, and to be able to predict, prevent and correct negative interactions.     

Recombinant Mycobacterium bovis BCG

The Bacillus Calmette–Guerin (BCG) is an attenuated strain of Mycobacterium bovis that has been broadly used as a vaccine against human tuberculosis. This live bacterial vaccine is able to establish a persistent infection and induces both cellular and humoral immune responses. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis of the potential use of this attenuated mycobacterium as a recombinant vaccine. Over the years, a range of strategies has been developed to allow controlled and stable expression of viral, bacterial and parasite antigens in BCG. Herein, we review the strategies developed to express heterologous antigens in BCG and the immune response elicited by recombinant BCG constructs. In addition, the use of recombinant BCG as an immunomodulator and future perspectives of BCG as a recombinant vaccine vector are discussed.     

Mycoplasma genitalium, an emerging sexually transmitted pathogen

Mycoplasma genitalium is a sexually transmitted organism associated with non-gonococcal urethritis in men and several inflammatory reproductive tract syndromes in women such as cervicitis, pelvic inflammatory disease, and infertility. There was evidence for an association of M. genitalium with endometritis and pelvic inflammatory disease (PID), but additional studies are necessary to confirm this. The evidence as to whether M. genitalium can cause adverse pregnancy outcomes such as preterm labor is conflicting. But the authors of some studies on M. genitalium as a cause of infertility have reported this association. This species is very difficult to culture; thus, nucleic acid amplification testing is the only method available for M. genitalium detection. The lack of a cell wall makes M. genitalium intrinsically resistant to antibiotics acting at this level, such as beta-lactams. The treatment of M. genitalium infections is not standardized. Macrolides are recommended, especially single-dose azithromycin; tetracyclines are responsible for a great number of therapeutic failures even no resistance mechanism has yet been demonstrated. Acquired resistance to macrolides and fluoroquinolones leading to therapeutic failure has also been reported. All this raises the issue of the most appropriate therapeutic management and requires drafting diagnostic and therapeutic guidelines for the treatment of M. genitalium infections.     

Vaccination with the Mycoplasma suis recombinant adhesion protein MSG1 elicits a strong immune response but fails to induce protection in pigs

Mycoplasma suis is the unculturable pathogen of porcine infectious anemia. The study was aimed to determine the immunogenicity and protective efficacy of MSG1, an immunodominant adhesin of M. suis as the first vaccine candidate against M. suis. The results demonstrated that recombinant MSG1 and Escherichia coli transformants expressing MSG1 (E. coli_MSG1) induced a strong humoral and cellular immunity against M. suis. The induced antibodies were found to be functionally active as confirmed by an in vitro adhesion inhibition assay. Both, IgG1 and IgG2 antibodies were induced, but E. coli_MSG1 immune response was characterized by a significantly higher IgG1 antibody production. Both vaccine candidates failed to protect against M. suis challenge. However, E. coli_MSG1 vaccination has a considerable effect on the severity of the disease as shown by higher post-challenge hemoglobin and hematocrit values in comparison to control groups. This indicated that a high IgG1 antibody titer is negatively connected with severity of M. suis-induced anemia. Furthermore, the induction of monospecific anti-MSG1 antibodies by both vaccine candidates clearly allows for the differentiation between infected and vaccinated animals (DIVA principle). Overall, the importance of MSG1 as potential vaccine candidate remains to be established. Future studies will evaluate the conditions (i.e. adjuvant, vaccination scheme, and application route) to optimize the effects of E. coli_MSG1 vaccines.     

Immunotherapy with combined DNA vaccines is an effective treatment for M. bovis infection in cattle

Mycobacterium bovis causes tuberculosis in cattle. Because current therapies inadequately prevent disease spread, new therapies for M. bovis infections are urgently needed. We treated M. bovis-infected cattle with combinations of DNA vaccines, resulting in significantly reduced pulmonary bacterial loads compared with the untreated group. Further, the pathological changes of lungs were also slight and the lesions were limited in animals treated with the combined DNA vaccines, whereas untreated animals had extensive lung fibrosis and consolidated lesions. This is the first report of DNA vaccine used to treat M. bovis. Thus, combined DNA vaccines may provide enhanced immunotherapeutic efficacy for treatment of bovine tuberculosis.     

Mycoplasma pneumoniae vaccine protective efficacy and adverse reactions—Systematic review and meta-analysis

Mycoplasma pneumoniae is a leading cause of both upper and lower respiratory infections that can lead to devastating sequela. Currently no primary prevention measures are available. During the 1960s and 1970s several inactivated M. pneumoniae vaccines were tested, some with encouraging results. Here we present a systematic review and meta-analysis on the efficacy and adverse effects of M. pneumoniae inactivated vaccines. Six clinical trials were found for efficacy calculation, with a total of 67,268 subjects. Vaccine associated adverse events were described in 15 studies. The summarized efficacy of M. pneumoniae vaccines against pneumonia regardless of etiologies was 36% (confidence interval (CI_95%) 25–45). The efficacy of the vaccines against M. pneumoniae associated pneumonia was 54% (35–67) or 42% (12–63) depending on diagnostic approach. Results were homogeneous without publication bias. No significant adverse reactions (including autoimmune effects) were observed. This study suggests that inactivated M. pneumoniae vaccines may reduce the total rates of both pneumonia and respiratory infections by ∼40%. We therefore suggest redeveloping M. pneumoniae vaccines, particularly for high-risk settings as well as in the general population.     

Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine

Mycoplasma hyopneumoniae causes severe economic losses to the swine industry worldwide and the prevention of its related disease, enzootic porcine pneumonia, remains a challenge. The P97 adhesin protein of M. hyopneumoniae should be a good candidate for the development of a subunit vaccine because antibodies produced against P97 could prevent the adhesion of the pathogen to the respiratory epithelial cells in vitro. In the present study, a P97 recombinant replication-defective adenovirus (rAdP97c) subunit vaccine efficiency was evaluated in pigs. The rAdP97c vaccine was found to induce both strong P97 specific humoral and cellular immune responses. The rAdP97c vaccinated pigs developed a lower amount of macroscopic lung lesions (18.5 ± 9.6%) compared to the unvaccinated and challenged animals (45.8 ± 11.5%). rAdP97c vaccine reduced significantly the severity of inflammatory response and the amount of M. hyopneumoniae in the respiratory tract. Furthermore, the average daily weight gain was slightly improved in the rAdP97c vaccinated pigs (0.672 ± 0.068 kg/day) compared to the unvaccinated and challenged animals (0.568 ± 0.104 kg/day). A bacterin-based commercial vaccine (Suvaxyn^® MH-one) was more efficient to induce a protective immune response than rAdP97c even if it did not evoke a P97 specific immune response. These results suggest that immunodominant antigens other than P97 adhesin are also important in the induction of a protective immune response and should be taken into account in the future development of M. hyopneumoniae subunit vaccines.     

Immunological responses and protective immunity in BCG vaccinated badgers following endobronchial infection with Mycobacterium bovis

European badgers (Meles meles) are a reservoir host of Mycobacterium bovis and are implicated in the transmission of tuberculosis to cattle in Ireland and Great Britain. The development of a vaccine for use in badgers is considered a key element of any campaign to eradicate the disease in livestock in both countries. In this study we have vaccinated groups of badgers with ∼5 × 10⁵ cfu of the BCG vaccine delivered via two alternative routes, subcutaneous and mucosal (intranasal/conjunctival). Following experimental endobronchial infection with ∼10⁴ cfu of M. bovis, all badgers were euthanised at 12 weeks post-infection. At post-mortem examination both vaccinated groups had significantly reduced severity of disease compared with the non-vaccinated controls. The analysis of immune responses throughout the study showed that vaccination with BCG did not generate any detectable immunological responses as measured by IFN-γ production in antigen-stimulated peripheral blood mononuclear cells (PBMC) and IgG serological responses. However, the levels of the responses increased following M. bovis infection, and the kinetic profiles corresponded to the severity of lesions recorded post-mortem. Significant differences were observed in the timing of development of the immune responses between vaccinates and controls. The results suggest that the immunological responses are associated with the levels of protective immunity and could be used as markers to monitor control of disease in badgers following vaccination.     

Local and systemic immune responses in pigs intramuscularly injected with an inactivated Mycoplasma hyopneumoniae vaccine

The immune response induced by intramuscular administration of a commercial inactivated Mycoplasma hyopneumonie whole-cell vaccine (Suvaxyn^®MH One) was investigated in conventional M. hyopneumoniae-free pigs. The animals were assigned randomly to two groups: non-vaccinated and vaccinated. Pigs in the vaccinated group were injected intramuscularly with the vaccine at 7 days of age, whereas non-vaccinated pigs received physiological saline solution (PBS). Pigs were euthanized and necropsied at 30, 36 and 58 days of age. Blood, bronchoalveolar lavage (BAL) fluid, spleen, lung and bronchial lymph nodes (BLN) were collected. Serum and BAL fluid were tested for the presence of antibodies by ELISA. Monomorphonuclear cells from the peripheral blood and tissues were isolated to quantify the T cell subsets by flow cytometry, and cytokine production by ELIspot and ELISA. Antibodies against M. hyopneumoniae were detected in serum of most vaccinated pigs at 30 days of age. M. hyopneumoniae specific IgG, IgM and IgA were detected in BAL fluid from vaccinated animals, but not from control animals. Significantly higher numbers of IL-12 secreting cells were observed in the lung at day 58 in the vaccinated than in the non-vaccinated group (p < 0.05). The number of IL-10 secreting cells from BLN was also higher in the vaccinated group at day 58 (p < 0.05). After restimulation in vitro, lymphocytes from BLN and lungs secreted significantly higher levels of IL-12 in the vaccinated group at day 58. These results show that the vaccine induced both systemic and mucosal cellular and humoral immune responses.     

Immune responses of a chimaeric protein vaccine containing Mycoplasma hyopneumoniae antigens and LTB against experimental M. hyopneumoniae infection in pigs

A recombinant chimaeric protein containing three Mycoplasma hyopneumoniae antigens (C-terminal portion of P97, heat shock protein P42, and NrdF) fused to an adjuvant, the B subunit of heat-labile enterotoxin of Escherichia coli (LTB), was used to immunize pigs against enzootic pneumonia. The systemic and local immune responses, as well as the efficacy of the chimaeric protein in inducing protection against experimental M. hyopneumoniae infection were evaluated. In total, 60 male piglets, purchased from a M. hyopneumoniae-free herd, at 4 weeks of age were randomly allocated to six different experimental groups of 10 animals each: recombinant chimaeric protein by intramuscular (IM) (1) or intranasal (IN) (2) administration, commercial bacterin by IM administration (3), and the adjuvant LTB by IM (4, control group A) or IN (5, control group B) administration. All groups were immunized at 24 and 38 days of age and challenged at 52 days of age. The sixth group that was not challenged was used as the negative control (IN [n = 5] or IM [n = 5] administration of the LTB adjuvant). Compared with the non-challenged group, administration of the chimaeric protein induced significant (P < 0.05) IgG and IgA responses against all individual antigens present in the chimaera, but it could not confer a significant protection against M. hyopneumoniae infection in pigs. This lack of effectiveness points towards the need for further studies to improve the efficacy of this subunit-based vaccine approach.     

Infection with a low virulent Mycoplasma hyopneumoniae isolate does not protect piglets against subsequent infection with a highly virulent M. hyopneumoniae isolate

The study aimed to evaluate the effect of an infection with low virulent isolates of M. hyopneumoniae (LV1 and LV2) on the subsequent infection with a highly virulent isolate (HV). Fifty-five, 3-week-old piglets free of M. hyopneumoniae were randomly allocated to 6 different groups. At 4 weeks of age (D0), groups LV1–HV and LV1 were intratracheally inoculated with LV1, groups LV2–HV and LV2 with LV2, and group HV with sterile culture medium. Four weeks later (D28), the pigs of these different groups were either intratracheally inoculated with the highly virulent isolate (groups LV1–HV, LV2–HV, HV) or with sterile culture medium (groups LV1 and LV2). A negative control group consisted of pigs inoculated with sterile culture medium on D0 and D28. All animals were necropsied at 28 days after the second inoculation (D56). Clinical symptoms were evaluated daily using a respiratory disease score (RDS). After necropsy, macroscopic and histopathological lung lesions were quantified and immunofluorescence (IF) testing on lung tissue and nested PCR on BAL fluid were performed for the detection of M. hyopneumoniae.     

First data on Eurasian wild boar response to oral immunization with BCG and challenge with a Mycobacterium bovis field strain

The Eurasian wild boar (Sus scrofa) is considered a reservoir for bovine tuberculosis (bTB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex in south-central Spain. The vaccination of wildlife with BCG offers an alternative to culling and to movement restriction for the control of bTB among wildlife reservoirs. In this study, we hypothesized that oral BCG immunization of wild boar would affect the expression of immunoregulatory genes and confer protection against M. bovis. Three groups were used to describe the infection, pathological findings and gene expression profiles in wild boar: BCG-vaccinated and M. bovis-challenged (vaccinated challenged group; N = 6), non-vaccinated and M. bovis-challenged (non-vaccinated challenged group; N = 4), and non-vaccinated and mock-infected (control group; N = 2) animals. M. bovis was isolated from 50% (3/6) and 75% (3/4) of vaccinated challenged and non-vaccinated challenged animals, respectively. All four wild boar from the non-vaccinated challenged group developed bTB-compatible lesions 114 days after challenge. In contrast, only 50% of vaccinated challenged wild boar developed lesions. The PBMC mRNA levels of IL4, RANTES, C3, IFN-gamma and methylmalonyl-CoA mutase (MUT) were analyzed at several days post-vaccination (dpi). When vaccinated challenged animals were compared to controls, all five genes were significantly upregulated at the time of M. bovis infection at 186 dpi but IFN-gamma levels were also upregulated at 11 and 46 dpi. The C3 and MUT mRNA levels were higher at 46 dpi, and 11 and 186 dpi, respectively, in vaccinated protected wild boar when compared to non-vaccinated challenged animals. At the end of the experiment (300 dpi), the mRNA levels of selected genes were lower in non-vaccinated challenged animals when compared to control wild boar. Exposing wild boar to a dose of 10⁴ cfu of M. bovis by the oropharyngeal route is an adequate protocol to produce an infection model in this species. Our results suggested that oral BCG immunization of wild boar results in the upregulation of immunoregulatory genes that may be associated with protective response to M. bovis infection in this species. More studies on vaccine efficacy, delivery, and safety will be needed to confirm if oral vaccination with BCG could be used in bTB control programs for reducing M. bovis infection and clinical disease in wild boar.     

Evaluation of recombinant Mycoplasma hyopneumoniae P97/P102 paralogs formulated with selected adjuvants as vaccines against mycoplasmal pneumonia in pigs

Pig responses to recombinant subunit vaccines containing fragments of eight multifunctional adhesins of the Mycoplasma hyopneumoniae (Mhp) P97/P102 paralog family formulated with Alhydrogel^® or Montanide™ Gel01 were compared with a commercial bacterin following experimental challenge. Pigs, vaccinated intramuscularly at 9, 12 and 15 weeks of age with either of the recombinant formulations (n = 10 per group) or Suvaxyn^® M. hyo (n = 12), were challenged with Mhp strain Hillcrest at 17 weeks of age. Unvaccinated, challenged pigs (n = 12) served as a control group. Coughing was assessed daily. Antigen-specific antibody responses were monitored by ELISA in serum and tracheobronchial lavage fluid (TBLF), while TBLF was also assayed for cytokine responses (ELISA) and bacterial load (qPCR). At slaughter, gross and histopathology of lungs were quantified and damage to epithelial cilia in the porcine trachea was evaluated by scanning electron microscopy. Suvaxyn^® M. hyo administration induced significant serological responses against Mhp strain 232 whole cell lysates (wcl) and recombinant antigen F3_P216, but not against the remaining vaccine subunit antigens. Alhydrogel^® and Montanide™ Gel01-adjuvanted antigen induced significant antigen-specific IgG responses, with the latter adjuvant eliciting comparable Mhp strain 232 wcl specific IgG responses to Suvaxyn^® M. hyo. No significant post-vaccination antigen-specific mucosal responses were detected with the recombinant vaccinates. Suvaxyn^® M. hyo was superior in reducing clinical signs, lung lesion severity and bacterial load but the recombinant formulations offered comparable protection against cilial damage. Lower IL-1β, TNF-α and IL-6 responses after challenge were associated with reduced lung lesion severity in Suvaxyn^® M. hyo vaccinates, while elevated pathology scores in recombinant vaccinates corresponded to cytokine levels that were similarly elevated as in unvaccinated pigs. This study highlights the need for continued research into protective antigens and vaccination strategies that will prevent Mhp colonisation and establishment of infection.     

Interaction of porcine circovirus type 2 and Mycoplasma hyopneumoniae vaccines on dually infected pigs

The objective of this study was to determine the effects of porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae vaccinations on disease severity in an experimental PCV2-M. hyopneumoniae dual challenge model. Vaccine effectiveness was evaluated using microbiological (PCV2 viremia and M. hyopneumoniae nasal shedding), immunological (neutralizing antibodies and interferon-γ-secreting cells), and pathological (gross lung lesions, histopathologic pulmonary and lymphoid lesions, and the presence of PCV2 antigen and M. hyopneumoniae DNA within the lesions) evaluations. Although M. hyopneumoniae potentiates the severity of PCV2-associated lesions and lesion-associated PCV2 antigen in dually challenged pigs, vaccination against M. hyopneumoniae alone did not reduce PCV2 viremia, PCV2-induced lesions, or PCV2 antigen in dually challenged pigs. In addition, vaccination against PCV2 did not reduce the nasal shedding of M. hyopneumoniae, the M. hyopneumoniae-induced pulmonary lesions or the lesion-associated M. hyopneumoniae DNA in dually challenged pigs. Dual challenge with PCV2 and M. hyopneumoniae did not interfere with the induction of active immunity induced by a previous single vaccination for either PCV2 or M. hyopneumoniae. The results of this study demonstrated that (i) vaccination against M. hyopneumoniae alone did not decrease the potentiation of PCV2-induced lesions by M. hyopneumoniae and (ii) vaccination against PCV2 alone decreased the potentiation of PCV2-induced lesions by M. hyopneumoniae in dually challenged pigs.     

Co-colonization by Haemophilus influenzae with Streptococcus pneumoniae enhances pneumococcal-specific antibody response in young children

Background

Streptococcus pneumoniae (Spn), Haemophilus influenzae (Hi) and Moraxella catarrhalis (Mcat) are common bacterial pathogens of respiratory infections and common commensal microbes in the human nasopharynx (NP). The effect of interactions among theses bacteria during co-colonization of the NP on the host immune response has not been evaluated. The objective of this study was to assess the impact of co-colonization by Hi or Mcat on the systemic antibody response to vaccine protein candidate antigens of Spn and similarly the impact of co-colonization by Spn and Mcat on antibody responses to Hi vaccine protein candidate antigens.

Methods

Serum samples were collected from healthy children at 6, 9, 15, 18, and 24 months of age when they were colonized with Spn, Hi, Mcat or their combinations. Quantitative ELISA was used to determine serum IgA and IgG against three Spn antigens and three Hi antigens, and as well as whole cells of non-typeable (NT) Spn and Hi.

Results

NP colonization by Spn increased serum IgA and IgG titers against Spn antigens PhtD, PcpA and PlyD and whole cells of NTSpn, and co-colonization of Hi or Mcat with Spn resulted in further increases of serum pneumococcal-specific antibody levels. NP colonization by Hi increased serum IgA and IgG titers against Hi antigens P6, Protein D and OMP26 and whole cells of NTHi, but co-colonization of Spn or Mcat with Hi did not result in further increase of serum NTHi-specific antibody levels.

Conclusion

Co-colonization of Hi or Mcat with Spn enhances serum antibody response to NTSpn whole cells and Spn vaccine candidate antigens PhtD, PcPA and PlyD1. Co-colonization appears to variably modulate pathogen species-specific host adaptive immune response.     

Impact of in vitro evolution on antigenic diversity of Mycobacterium bovis bacillus Calmette-Guerin (BCG)

Mycobacterium bovis bacillus Calmette-Guerin (BCG), the only vaccine currently used against tuberculosis, is an attenuated derivative of M. bovis that has been propagated in vitro for more than 40 years. We have previously reported that the experimentally-verified human T cell epitopes of the M. tuberculosis complex (MTBC) are the most conserved elements of the genome; whether immune recognition is the force driving the conservation of epitopes in the MTBC is unknown. Therefore, we sequenced the genomes of 12 BCG strains to determine whether T cell epitopes were under selection pressure during BCG in vitro evolution. We constructed a genome-wide phylogeny and refined the previously-determined BCG phylogeny. Notably, we identified a new cluster between BCG Japan and BCG Russia, and repositioned the relationships of several strains within the lineage. We also compared the sequence diversity of 1530 experimentally verified human T cell epitopes in the BCG vaccines with those in the MTBC. We found 23% of the known T cell epitopes are absent, and that the majority (82%) of the absent epitopes in BCG are contained in 6 proteins encoded in 2 regions of difference (RD) unique to BCG strains. We also found that T cell epitope sequences in BCG are more conserved than non-epitope sequences in the same gene. Finally, we find evidence that epitope sequence variation in BCG potentially affects human T cell recognition. These findings provide new insight into sequence variation in a slow-growing bacterium closely related to the MTBC that has been subjected to prolonged passage outside of a mammalian host, and indicate little difference in the extent of variation in vivo and in vitro.