It was shown recently that Mycobacterium tuberculosis expresses five proteins that are homologous to Rpf (resuscitation promoting factor), which is secreted by growing cells of Micrococcus luteus. Rpf is required to resuscitate the growth of dormant Micrococcus luteus organisms, and its homologues may be involved in mycobacterial reactivation. Mycobacterial Rpf-like products are secreted proteins, which makes them candidates for recognition by the host immune system and anti-Rpf immune responses potentially protective against reactivated tuberculosis. Here we report that the Rpf protein itself and four out of five of its mycobacterial homologues, which were administered as subunit vaccines to C57BL/6 mice, are highly immunogenic. Rpf-like proteins elicit immunoglobulin G1 (IgG1) Tuberculosis (TB) remains one of the most important causes of morbidity and mortality worldwide (8,10,38,39), and this situation dictates an urgent need for improved measures for controlling TB. The increasing numbers of multidrug-resistant TB cases (6, 37) suggest that the development of innovative vaccine strategies is, perhaps, a method of choice for controlling the spread of TB. Mycobacterium bovis BCG (attenuated M. bovis strain) represents the only vaccine available against TB as yet, although its efficacy in well-controlled clinical trials appears to be highly varied (9,13,14). Importantly, it is very likely that BCG vaccination does not protect against adult pulmonary TB in areas where TB is endemic (9, 19), i.e., the vaccine's effect is negligible exactly where it is most needed. An elegant, recent study of mice provided a rationale for the low efficacy of BCG in the regions where there is a high level of exposure to saprophytic mycobacteria (7). A varied BCG performance, as well as the obvious problem of using a live BCG vaccine in populations experiencing a substantial increase in the spread of human immunodeficiency virus (15, 33), validates the development of anti-TB vaccines whose efficacy is not dependent upon the persistence of live mycobacteria in the host.Among several strategies to replace BCG with novel TB vaccine candidates, e.g., vaccination with a subunit protein, naked DNA, and improved whole bacterial vaccines (for a review, see reference 20), vaccination with a subunit protein is the approach best characterized for animal models (12,17,29).

We previously demonstrated that mice of the I/St strain are extremely susceptible to Mycobacterium tuberculosis, as well as to the taxonomically distant intracellular bacteria Chlamydia pneumoniae and Salmonella enterica. To broaden our knowledge about the control of susceptibility to intracellular pathogens, we studied the infection caused by Mycobacterium avium virulent strain 724 in a panel of inbred mouse strains and found that I/St mice are resistant to M. avium. By comparing I/St mice with B6 mice, we demonstrated that (i) B6 mice are much more susceptible to infection caused by M. avium in terms of bacterial multiplication in the lung tissue and severity of lung pathology; (ii) in B6 mice but not in I/St mice infection leads to prolonged leukocyte infiltration of the lung tissue, development of necrotic lung granulomata, and lethality; and (iii) the unfavorable infectious course in B6 mice is accompanied by elevated production of gamma interferon, tumor necrosis factor alpha, and especially interleukin-12 in the lungs. Importantly, M. avium-resistant I/St mice carry a functional r allele of the Slc11a1 (formerly Nramp1) gene, while B6 mice have the Slc11a1 s genotype. Segregation genetic analysis of (I/St × B6) F2 hybrids demonstrated that susceptibility or resistance to infection caused by M. avium largely depended upon the Slc11a1 genotype and that other genetic traits had a relatively weak influence. This close-to-monogenic pattern differs sharply from the host control of many other intracellular bacterial infections, for which the involvement of numerous quantitative trait loci has been ubiquitously observed.

Most viruses are known to spontaneously generate defective viral genomes (DVG) due to errors during replication. These DVGs are subgenomic and contain deletions that render them unable to complete a full replication cycle in the absence of a co-infecting, non-defective helper virus. DVGs, especially of the copyback type, frequently observed with paramyxoviruses, have been recognized to be important triggers of the antiviral innate immune response. DVGs have therefore gained interest for their potential to alter the attenuation and immunogenicity of vaccines. To investigate this potential, accurate identification and quantification of DVGs is essential. Conventional methods, such as RT-PCR, are labor intensive and will only detect primer sequence-specific species. High throughput sequencing (HTS) is much better suited for this undertaking. Here, we present an HTS-based algorithm called DVG-profiler to identify and quantify all DVG sequences in an HTS data set generated from a virus preparation. DVG-profiler identifies DVG breakpoints relative to a reference genome and reports the directionality of each segment from within the same read. The specificity and sensitivity of the algorithm was assessed using both in silico data sets as well as HTS data obtained from parainfluenza virus 5, Sendai virus and mumps virus preparations. HTS data from the latter were also compared with conventional RT-PCR data and with data obtained using an alternative algorithm. The data presented here demonstrate the high specificity, sensitivity, and robustness of DVG-profiler. This algorithm was implemented within an open source cloud-based computing environment for analyzing HTS data. DVG-profiler might prove valuable not only in basic virus research but also in monitoring live attenuated vaccines for DVG content and to assure vaccine lot to lot consistency.

Complete genomic sequences of a non-redundant set of 70 recombinants between three serotypes of attenuated Sabin polioviruses as well as location (based on partial sequencing) of crossover sites of 28 additional recombinants were determined and compared with the previously published data. It is demonstrated that the genomes of Sabin viruses contain distinct strain-specific segments that are eliminated by recombination. The presumed low fitness of these segments could be linked to mutations acquired upon derivation of the vaccine strains and/or may have been present in wild-type parents of Sabin viruses. These “weak” segments contribute to the propensity of these viruses to recombine with each other and with other enteroviruses as well as determine the choice of crossover sites. The knowledge of location of such segments opens additional possibilities for the design of more genetically stable and/or more attenuated variants, i.e., candidates for new oral polio vaccines. The results also suggest that the genome of wild polioviruses, and, by generalization, of other RNA viruses, may harbor hidden low-fitness segments that can be readily eliminated only by recombination.

Sabin-strain oral polio vaccines (OPV) can, in rare instances, cause disease in recipients and susceptible contacts or evolve to become circulating vaccine-derived strains with the potential to cause outbreaks. Two novel type 2 OPV (nOPV2) candidates were designed to stabilize the genome against the rapid reversion that is observed following vaccination with Sabin OPV type 2 (mOPV2). Next-generation sequencing and a modified transgenic mouse neurovirulence test were applied to shed nOPV2 viruses from phase 1 and 2 studies and shed mOPV2 from a phase 4 study. The shed mOPV2 rapidly reverted in the primary attenuation site (domain V) and increased in virulence. In contrast, the shed nOPV2 viruses showed no evidence of reversion in domain V and limited or no increase in neurovirulence in mice. Based on these results and prior published data on safety, immunogenicity, and shedding, the nOPV2 viruses are promising alternatives to mOPV2 for outbreak responses.

No abstract

Virus growth during influenza vaccine manufacture can lead to mutations that alter antigenic properties of the virus, and thus may affect protective potency of the vaccine. Different reassortants of pandemic "swine" H1N1 influenza A vaccine (121XP, X-179A and X-181) viruses as well as wild type A/California/07/2009(H1N1) and A/PR/8/34 strains were propagated in embryonated eggs and used for DNA/RNA Illumina HiSeq and MiSeq sequencing. The RNA sequences of these viruses published in NCBI were used as references for alignment of the sequencing reads generated in this study. Consensus sequences of these viruses differed from the NCBI-deposited sequences at several nucleotides. 121XP stock derived by reverse genetics was more heterogeneous than X-179A and X-181 stocks prepared by conventional reassortant technology. Passaged 121XP virus contained four non-synonymous mutations in the HA gene. One of these mutations (Lys226Glu) was located in the Ca antigenic site of HA (present in 18% of the population). Two non-synonymous mutations were present in HA of viruses derived from X-179A: Pro314Gln (18%) and Asn146Asp (78%). The latter mutation located in the Sa antigenic site was also detected at a low level (11%) in the wild-type A/California/07/2009(H1N1) virus, and was present as a complete substitution in X-181 viruses derived from X-179A virus. In the passaged X-181 viruses, two mutations emerged in HA: a silent mutation A1398G (31%) in one batch and G756T (Glu252Asp, 47%) in another batch. The latter mutation was located in the conservative region of the antigenic site Ca. The protocol for RNA sequencing was found to be robust, reproducible, and suitable for monitoring genetic consistency of influenza vaccine seed stocks.

Genetic variation in the major histocompatibility complex (MHC) influences susceptibility and immune responses to Mycobacterium tuberculosis in mice and humans, but connections among the severity of tuberculosis (TB), dynamic changes in T cell responses to mycobacteria, and MHC genetic polymorphisms are poorly characterized. The overall effect of the MHC genes on TB susceptibility and cellular responses to mycobacteria is moderate; thus, such studies provide reliable results only if congenic mouse strains bearing a variety of H2 haplotypes on an identical genetic background are analyzed. Using a panel of H2-congenic strains on the B10 background, we demonstrate that T cells from mice of three different strains, which are resistant to TB infection, readily respond by proliferation to repeated stimulations with mycobacterial sonicate, whereas T cells from three susceptible mouse strains die after the second stimulation with antigen. This difference is specific, as T cells from TB-susceptible and -resistant mouse strains do not differ in response to irrelevant antigens. The CD4/CD8 ratio in immune lymph nodes correlates strongly and inversely with TB susceptibility, being significantly lower in resistant mice as a result of an increased content of CD8+ cells. These differences between the two sets of mouse strains correlate with an elevated level of activation-induced T cell apoptosis in TB-susceptible mice and a higher proportion of activated CD44+ CD62 ligand- T cells in TB-resistant mice. These results may shed some light on the nature of the cellular basis of MHC-linked differences in susceptibility to TB.