Potentially conflicting selective forces that shape the vls antigenic variation system in Borrelia burgdorferi

Changing environmental conditions present an evolutionary challenge for all organisms. The environment of microbial pathogens, including the adaptive immune responses of the infected host, changes rapidly and is lethal to the pathogen lineages that cannot quickly adapt. The dynamic immune environment creates strong selective pressures favoring microbial pathogen lineages with antigenic variation systems that maximize the antigenic divergence among expressed antigenic variants. However, divergence among expressed antigens may be constrained by other molecular features such as the efficient expression of functional proteins. We computationally examined potential conflicting selection pressures on antigenic variation systems using the vls antigenic variation system in Borrelia burgdorferi as a model system. The vls system alters the sequence of the expressed antigen by recombining gene fragments from unexpressed but divergent ‘cassettes’ into the expression site, vlsE. The in silico analysis of natural and altered cassettes from seven lineages in the B. burgdorferi sensu lato species complex revealed that sites that are polymorphic among unexpressed cassettes, as well as the insertion/deletion mutations, are organized to maximize divergence among the expressed antigens within the constraints of translational ability and high translational efficiency. This study provides empirical evidence that conflicting selection pressures on antigenic variation systems can limit the potential antigenic divergence in order to maintain proper molecular function.     

Inferring the antigenic epitopes for highly pathogenic avian influenza H5N1 viruses

The evasion of influenza virus from host immune surveillance is mainly mediated through its surface protein hemagglutinin (HA), the main component of influenza vaccine. Thus, identification of influenza virus antigenic epitopes on HA can not only help us understand the molecular mechanisms of viral immune escape but also facilitate vaccine strain selection. Despite previous efforts, there is a lack of systematic definition of the antigenic epitopes for the highly pathogenic avian influenza (HPAI) H5N1 viruses. In this study, we infer the HA antigenic epitopes for H5N1 viruses by integrating the antigenic sites mapped from the HA of human influenza H3N2 viruses, the sites which were reported to be associated with immune escape in H5 viruses and the mutation hotspot sites identified in the evolutionary history of HPAI H5N1 viruses. We show that these inferred antigenic epitopes play significant roles in antigenic variation of HPAI H5N1 viruses. Based on inferred antigenic epitopes, we further develop a computational method to effectively predict antigenic variants for HPAI H5N1 viruses (available at http://biocloud.hnu.edu.cn/predict/html/index.html). Therefore, our work has not only inferred the antigenic epitopes for HPAI H5N1 viruses but also provided an effective computational method to assist vaccine recommendations for protection against the deadly bird flu.     

On the need for, and the delivery of, cross-protective vaccines

The rhinoviruses that are instrumental in causing about one-third of the outbreaks of the common cold present us with some 100 or so serotypes whose convalescent sera do not cross-neutralise. A similar situation prevails with the organism that causes gonorrhoea. Both the HIV and the protozoan causing malaria are notorious for their ability to evade the immune system by changes to their antigenic profile. Similarly, we face continual changes in the antigenic determinants of the influenza virus. It is clear that we require vaccine for these diseases that provide protection against a wide variety of basic variants. This can be achieved, as was shown by Arvind Kumar, who, in his PhD project, generated monoclonal antibodies to cross-reacting yet neutralising epitopes of a number of rhinoviruses. Such antibodies also neutralised some Coxsackie viruses as well as some of the types of Poliovirus. This demonstration of feasibility will be explored further in my paper with a view to arriving at a general approach to the production of vaccines whose humoral and cellular responses can neutralise a wide cross-section of serotype variants.     

Cloning and expression of recombinant flagellar protein flaB from Borrelia burgdorferi

OBJECTIVE: Lyme borreliosis is an arthropod transmitted infection caused by some species of the Borrelia genus. Current diagnosis employs serological testing and detection of Borrelia-specific antibodies. Using recombinant Borrelia burgdorferi antigens may improve assay specificity and sensitivity. One of the immunodominant Borrelia antigens that elicit a strong and early immune response is FlaB, making it appropriate for recombinant protein based serological diagnostic tests. MATERIAL AND METHODS: Borrelia burgdorferi genomic DNA was isolated and used as a template for the amplification of the flaB gene. The gene was cloned in the expression vector pGEX-2T. RESULTS: The amplified flaB gene was cloned in the expression vector yielding a GST-FlaB fusion gene. The gene ligated in-frame was expressed as the recombinant GST-FlaB protein. After visualization by polyacrylamide gel electrophoresis the successful expression of the FlaB protein was confirmed by immunoblotting. CONCLUSION: The expression and purification of the recombinant FlaB protein is a prerequisite for obtaining large amounts of the product through a simple and labour-free procedure, which will facilitate the diagnosis of Lyme disease.     

Antigenic heterogeneity within influenza A (H3N2) virus strains

On the basis of their antigenic properties, influenza virus strains are classified into types and subtypes, which are further subdivided into variants that differ to various degrees in haemagglutination-inhibition assays. Evidence is presented that during infection with an influenza A(H3N2) virus the respiratory tract of a human patient often harbours more than one antigenic virus variant. These variants are frequently propagated by embryonated fowl eggs and monkey cells with different efficiencies, and this may lead to the selection of different variants by either of these host systems. Also, passage of virus by a given host is sometimes attended by changes in reactivity in haemagglutination-inhibition tests. In some cases the heterogeneity described also affects the specific immunogenicity of the virus in ferrets. Virus strains cloned in monkey kidney cell cultures gave variants that were stable upon further passage. These results may have implications for antigenic and biochemical investigations of epidemiologically relevant virus variants. It is argued that the antigenic drift of influenza A(H3N2) viruses is best characterized by analyses, both with post-infection ferret antisera and with panels of monoclonal antibodies, of virus strains isolated and passaged in monkey kidney cell cultures only.     

Source for influenza pandemics

There are three ways how influenza A viruses can escape the immune response in the human population: (1) By antigenic drift. This means by mutation and selection of variants under the selection pressure of the immune system. These variants have amino acid replacements mainly in the epitopes of the hemagglutinin. (2) By antigenic shift. This means replacement of at least the hemagglutinin gene of the prevailing human strain by the allelic gene of an avian influenza virus by reassortment. (3) As a rare event, direct or indirect introduction of an avian influenza virus in toto into the human population. A prior introduction of an avian virus into pigs and an adaptation to the new host might be a presupposition for its final passage to humans. In this sense the nowadays situation is reminiscent to that of about 100 years ago, when an avian virus was presumably first introduced into pigs, and from there into humans. Immediately or some time thereafter the disastrous Spanish Flu in 1918/19 had killed at least 20,000,000 people in one winter. Pandemic strains can be created by all three means, however the most common way is by reassortment. In order to recognize a pandemic strain as soon as possible a worldwide surveillance system and collaborating laboratories equipped with corresponding modern technologies are required.     

Identification and characterization of Borrelia antigens as potential vaccine candidates against Lyme borreliosis

The three Borrelia species, Borrelia afzelii, Borrelia burgdorferi and Borrelia garinii are the main species causing the most common tick-borne zoonosis, Lyme borreliosis. By applying a genomic approach relying on human antibodies we have identified 122 antigenic Borrelia proteins associated with Lyme borreliosis, including already known and published protective antigens. The heterogeneity of the Borrelia species causing Lyme borreliosis makes the search for conserved antigens providing broad protection challenging. Using several in vitro assays we narrowed down the selection to 15 vaccine candidates. These antigens were further analyzed for antigenicity and cross-reactivity using sera from mice infected with the three pathogenic Borrelia species. All antigens analyzed showed a high degree of cross-reactivity between the three Borrelia species, essential for providing cross-protection. We also investigated whether mice infected with B. afzelii through tick exposure are primed to mount cytokine responses. For a selection of these antigens, we observed preferentially a pro-inflammatory response in C3H/HeN mice, while in contrast also a type 2 T cell response was seen in the Borrelia-resistant mouse strain BALB/c. Thus, antigens mounting a type 2 or mixed type 2/type 1 T cell response might be preferred vaccine candidates for evaluation in animal models of Lyme borreliosis.     

Molecular typing of canine parvovirus strains circulating from 2008 to 2012 in an organized kennel in India reveals the possibility of vaccination failure

Canine parvovirus-2 (CPV-2), which emerged in 1978, is considered as the major viral enteric pathogen of the canine population. With the emergence of new antigenic variants and incidences of vaccine failure, CPV has become one of the dreaded diseases of the canines worldwide. The present study was undertaken in an organized kennel from North India to ascertain the molecular basis of the CPV outbreaks in the vaccinated dogs. 415 samples were collected over a 5 year period (2008–2012). The outbreak of the disease was more severe in 2012 with high incidence of mortality in pups with pronounced clinical symptoms. Molecular typing based on the VP2 gene was carried out with the 11 isolates from different years and compared with the CPV prototype and the vaccine strains. All the isolates in the study were either new CPV-2a (2012 isolates) or new CPV-2b (2008 and 2011 isolates). There were amino acid mutations at the Tyr324Ile and at the Thr440Ala position in five isolates from 2012 indicating new CPV mutants spreading in India. The CPV vaccines used in the present study failed to generate protective antibody titer against heterogeneous CPV antigenic types. The findings were confirmed when the affected pups were treated with hyper-immune heterogeneous purified immunoglobulin’s against CPV in dogs of different antigenic types.     

Epitopes of the human malaria parasite P. falciparum carried on the surface of HBsAg particles elicit an immune response against the parasite.

The development of recombinant subunit vaccines against pathogenic organisms requires not only the identification of epitopes eliciting a protective immune response but also suitable carriers with adjuvant function. B- and T-cell epitopes of the malaria vaccine candidate gp190 were selected on the basis of a systematic search along the gp190 molecule and by computer prediction based on the amino acid sequence. Using some of the epitopes identified, we have redesigned the surface of the hepatitis B surface antigen lipoprotein particles by replacing the major antigenic determinants with malaria-specific sequences of up to 61 amino acids in length. Upon expression via vaccinia virus the hybrid particles elicit an anti-gp190 immune response in animals. Monoclonal antibodies derived from such infections recognize the native parasite.     

Deceptive imprinting and immune refocusing in vaccine design

A large number of the world's most widespread and problematic pathogens evade host immune responses by inducing strain-specific immunity to immunodominant epitopes with high mutation rates capable of altering antigenic profiles. The immune system appears to be decoyed into reacting to these immunodominant epitopes that offer little cross protection between serotypes or subtypes. For example, during HIV-1 infection, the immune system reacts strongly to the V1, V2, and/or V3 loops of the surface envelope glycoprotein but not to epitopes that afford broad protection against strain variants. Similarly, the host mounts strain-specific immunity to immunodominant epitopes of the influenza hemagglutinin (HA) protein. A large number of pathogens appear to exploit this weakness in the host immune system by focusing antigenic attention upon highly variable epitopes while avoiding surveillance toward more highly conserved receptor binding sites or other essential functional domains. Because the propensity of the immune system to react against immunodominant strain-specific epitopes appears to be genetically hard-wired, the phenomenon has been termed "deceptive imprinting." In this review, the authors describe observations related to deceptive imprinting in multiple systems and propose strategies for overcoming this phenomenon in the design of vaccines capable of inducing protection against highly variable pathogens.     

Immunological assessment of plant-derived avian flu H5/HA1 variants

Polypeptide variants of the HA1 antigenic domain of the H5N1 avian influenza virus hemagglutinin (HA) molecule were produced in plants using transient and stable expression systems and fused with His/c-myc tags or with mouse or human Fc antibody fragments. The resulting peptides were purified and used for intramuscular immunization of mice. While the recombinant HA1 variants induced a significant serum humoral immune response in the mice, none of the HA1 preparations induced virus-neutralizing antibodies. Fusion with the Fc fragment improved overall yield of the constructs and allowed purification requiring only a single step, but led to no detectable fusion-related enhancement of immunogenicity or quality of immune response.     

Questions on causality and responsibility arising from an outbreak of Pseudomonas aeruginosa infections in Norway

In 2002, Norway experienced a large outbreak of Pseudomonas aeruginosa infections in hospitals with 231 confirmed cases. This fuelled intense public and professional debates on what were the causes and who were responsible. In epidemiology, other sciences, in philosophy and in law there is a long tradition of discussing the concept of causality. We use this outbreak as a case; apply various theories of causality from different disciplines to discuss the roles and responsibilities of some of the parties involved. Mackie's concept of INUS conditions, Hill's nine viewpoints to study association for claiming causation, deterministic and probabilistic ways of reasoning, all shed light on the issues of causality in this outbreak. Moreover, applying legal theories of causation (counterfactual reasoning and the "but-for" test and the NESS test) proved especially useful, but the case also illustrated the weaknesses of the various theories of causation. We conclude that many factors contributed to causing the outbreak, but that contamination of a medical device in the production facility was the major necessary condition. The reuse of the medical device in hospitals contributed primarily to the size of the outbreak. The unintended error by its producer – and to a minor extent by the hospital practice – was mainly due to non-application of relevant knowledge and skills, and appears to constitute professional negligence. Due to criminal procedure laws and other factors outside the discourse of causality, no one was criminally charged for the outbreak which caused much suffering and shortening the life of at least 34 people.     

Egg-grown and tissue-culture-grown variants of influenza A (H3N2) virus with special attention to their use as antigens in seroepidemiology

A field strain of influenza A (H3N2) virus isolated in embryonated eggs during the 1984-5 influenza outbreak (A/Finland/13/85E) was compared in an antigenic analysis with virus from the same clinical specimen isolated in MDCK cell cultures (A/Finland/13/85M). The M-virus appeared to be more sensitive to haemagglutination-inhibiting antibodies against heterologous viruses than did the E-virus. The results of propagation and plaque purification experiments support the hypothesis that a single clinical specimen may consist of distinct antigenic variant subpopulations promoted selectively by the host during isolation procedures. Receptor-binding properties are discussed as a possible explanation for this selectivity. A set of 471 paired sera consisting of pre-epidemic and post-epidemic specimens taken from the same subjects in 1984-5 was studied for haemagglutination-inhibiting antibodies to six influenza A (H3N2) virus strains, including the E-virus and the M-virus from A/Finland/13/85. Of the antigens used, the M-virus detected significant antibody increases more frequently than did the E-virus (10.0 v. 5.9%). The superiority of the M-virus may rest primarily in its ability to pick out anamnestic antibody responses. Irrespective of this cross-reactivity, pre-epidemic antibody to the M-virus was fairly well associated with protection. In the set of sera (230 specimens) collected in summer 1985 to represent different age groups, the antibody status against the M-virus was significantly better than the status against the E-virus. The results suggest that, at least in some instances, antibody to MDCK-grown virus is a more accurate indicator of the immune status of a community than antibodies to egg-grown virus variants.     

Variation among strains of Corynebacterium diphtheriae during an outbreak in a restricted environment

Strains of Corynebacterium diphtheriae isolated from a small outbreak in the restricted environment of a Mental Hospital were examined. All belonged to one serotype, but there was marked variation in diphthericin type, in sensitivity to bacteriophages and in the minor antigens possessed. One strain was non-virulent and laboratory-produced variants of this non-virulent strain showed changes in some of the characteristics used in the identification and typing of the organism, such as diphthericin type, sensitivity to bacteriophages and diphthericins, virulence, starch fermentation and, to a lesser extent, in antigenic structure. The epidemiological and experimental findings are consistent with the hypothesis that the strains isolated, both in the hospital and in the laboratory, were derivatives of a single parent and the mechanism of some of the variations could be related to changes in some structural component such as the cell membrane or the cell wall.     

Typing African relapsing fever spirochetes

Relapsing fever Borrelia spp. challenge microbiologic typing because they possess segmented genomes that maintain essential genes on large linear plasmids. Antigenic variation further complicates typing. Intergenic spacer (IGS, between 16S-23S genes) heterogeneity provides resolution among Lyme disease-associated and some relapsing fever spirochetes. We used an IGS fragment for typing East African relapsing fever Borrelia spp. Borrelia recurrentis and their louse vectors showed 2 sequence types, while 4 B. duttonii and their tick vectors had 4 types. IGS typing was unable to discriminate between the tick- and louseborne forms of disease. B. crocidurae, also present in Africa, was clearly resolved from the B. recurrentis/B. duttonii complex. IGS analysis of ticks showed relapsing fever Borrelia spp. and a unique clade, distant from those associated with relapsing fever, possibly equivalent to a novel species in ticks from this region. Clinical significance of this spirochete is undetermined.     

Polymorphism in the subtelomeric regions of chromosomes of Kinetoplastida

Leishmania spp. and the related kinetoplastid Trypanosoma brucei are single-celled parasites. In Leishmania, the nuclear genome comprises 36 diploid chromosomes and occasional amplified minichromosomes, while the T. brucei nucleus contains 11 larger diploid chromosomes and a variable number of intermediate-sized and minichromosomes. This paper primarily describes the subtelomeric structure of the larger diploid chromosomes of L. major and T. brucei, although some aspects may also apply to smaller chromosomes. The diploid chromosomes contain most protein-coding genes and vary in size. The telomeric sequence is common to both species, but adjacent subtelomeric repeats vary between species and chromosomes. It is possible that some of the complex repeats described here play a role in stabilizing replication and copy number of the chromosomes. The subtelomeric regions of T. brucei chromosomes differ from those of other protozoan parasites, as they are dedicated to expression sites for variant surface glycoprotein genes, used by the parasite to evade immune destruction by antigenic variation. Variation in these sites creates segmental aneuploidy in many T. brucei chromosomes.     

Hierarchical modeling of melanocortin 1 receptor variants with skin cancer risk

The human MC1R gene is highly polymorphic among lightly pigmented populations, and several variants in the MC1R gene have been associated with increased risk of both melanoma and nonmelanoma skin cancers. The functional consequences of MC1R gene variants have been studied in vitro and in vivo in postulated causal pathways, such as G‐protein‐coupled signaling transduction, pigmentation, immune response, inflammatory response, cell proliferation, and extracellular matrix adhesion. In a case‐control study nested within the Nurses’ Health Study, we utilized hierarchical modeling approaches, incorporating quantitative information from these functional studies, to examine the association between particular MC1R alleles and the risk of skin cancers. Different prior matrices were constructed according to the phenotypic associations in controls, cell surface expression, and enzymatic kinetics. Our results showed the parameter variance estimates of each single nucleotide polymorphism (SNP) were smaller when using a hierarchical modeling approach compared to standard multivariable regression. Estimates of second‐level parameters gave information about the relative importance of MC1R effects on different pathways, and odds ratio estimates changed depending on prior models (e.g., the change ranged from ?21% to 7% for melanoma risk assessment). In addition, the estimates of prior model hyperparameters in the hierarchical modeling approach allow us to determine the relevance of individual pathways on the risk of each of the skin cancer types. In conclusion, hierarchical modeling provides a useful analytic approach in addition to the widely used conventional models in genetic association studies that can incorporate measures of allelic function.     

The study molecular epidemiology of canine parvovirus, Europe

Canine parvovirus (CPV), which causes hemorrhagic enteritis in dogs, has 3 antigenic variants: types 2a, 2b, and 2c. Molecular method assessment of the distribution of the CPV variants in Europe showed that the new variant CPV-2c is widespread in Europe and that the viruses are distributed in different countries.     

Cloning, expression and evaluation of the efficacy of a recombinant Eimeria tenella sporozoite antigen in birds

Eimeria infection in poultry is of significant economic interest worldwide. Development of a cost-effective sub-unit vaccine that provides cross-protection may help reduce loss in poultry industry. One approach explored by many investigators is to block the parasite invasion into gut epithelium. Use of microneme proteins to prevent parasite invasion is one of the most straightforward approaches in developing a preventive vaccine. Here we describe cloning and expression of microneme-1 protein of Eimeria tenella, obtained from an outbreak sample from India. We have evaluated the ability of the recombinant protein to elicit both cell mediated immune (CMI) and humoral immune responses. We also evaluated the efficacy of the recombinant protein in protecting against a homologous challenge. Our data indicate recombinant EtMIC1 is able to impart partial protection against homologous challenge in chicken. Inclusion of more invasion proteins may improve the efficacy of prophylactic vaccine against Coccidiosis.