Mark S. Hanson

Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi

The genome of the bacterium Borrelia burgdorferi B31, the aetiologic agent of Lyme disease, contains a linear chromosome of 910,725 base pairs and at least 17 linear and circular plasmids with a combined size of more than 533,000 base pairs. The chromosome contains 853 genes encoding a basic set of proteins for DNA replication, transcription, translation, solute transport and energy metabolism, but, like Mycoplasma genitalium, it contains no genes for cellular biosynthetic reactions. Because B. burgdorferi and M. genitalium are distantly related eubacteria, we suggest that their limited metabolic capacities reflect convergent evolution by gene loss from more metabolically competent progenitors. Of 430 genes on 11 plasmids, most have no known biological function; 39% of plasmid genes are paralogues that form 47 gene families. The biological significance of the multiple plasmid-encoded genes is not clear, although they may be involved in antigenic variation or immune evasion.

Use of a Whole Genome Approach To Identify Vaccine Molecules Affording Protection against Streptococcus pneumoniae Infection

ABSTRACT Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen Streptococcus pneumoniae, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated S. pneumoniaeinfection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against S. pneumoniae.

Safety and immunogenicity of recombinant Bacille Calmette-Guérin (rBCG) expressing Borrelia burgdorferi outer surface protein A (OspA) lipoprotein in adult volunteers: a candidate Lyme disease vaccine.

This phase I clinical trial was designed to determine the feasibility of using rBCG as a live bacterial vaccine vector for the outer surface protein A (OspA) of Borrelia burgdorferi and as model for other vaccines based on a rBCG vector. To construct the vaccine, a signal peptide derived from a mycobacterial lipoprotein was used to direct the export, and membrane-associated surface expression, of OspA in a standard strain of BCG (Connaught). The rBCG OspA vaccine was safe and immunogenic in several animal species, and protective in a mouse model of Lyme borreliosis. An intradermal injection (0.1 ml) of rBCG OspA was administered to 24 healthy adult volunteers sequentially at one of four dose levels, ranging from 2.0 x 10(4) CFU to 2 x 10(7) CFU, using a dose-escalation design. All volunteers were initially PPD-skin test and OspA antibody negative, and they were monitored for 2 years after immunization. Three volunteers had mild flu-like reactions 1-2 days after vaccination. Local ulceration and drainage at the site of injection, which occurred in 50% and 83% of volunteers in the two highest dose groups, persisted for 1-70 days before the ulcers healed. Most of the drainage samples yielded rBCG colonies that contained the OspA plasmid. Thirteen of 24 vaccinees, principally in the two highest dose groups, converted their PPD skin tests from negative to positive. None of the 24 volunteers developed OspA antibody. In conclusion, the current rBCG vaccine construct, the first such construct tested in humans, had a safety profile comparable to that of licensed BCG, but it did not elicit primary humoral responses to the vectored antigen.

Lyme disease vaccines

Lyme disease is the most common tick-borne disease in the United States; 16 455 cases were reported in 1996 [1], and the actual incidence may be 10-fold higher [2]. In areas with a high incidence of Lyme disease, the licensing of vaccines against the causative spirochete, Borrelia burgdorferi, has been anticipated with great enthusiasm by the medical community and the general public. Two recombinant vaccines, each containing 30 g of outer surface protein A (OspA), are in the final stages of approval by the U.S. Food and Drug Administration (FDA). The SmithKline Beecham product (LYMErix) contains an aluminum adjuvant, whereas the Pasteur Merieux Connaught product (Imulyme) does not. In studies in adults, 96% of participants showed a fourfold increase in antibodies against OspA antigen 2 weeks after receiving a second dose of either vaccine [3], although clinical trials have indicated that a third dose is necessary to further boost antibody levels and provide a protective efficacy of more than 80%. Diminished efficacy was noted in persons older than 65 years of age [4, 5]. The vaccines protect in a novel manner. Animal studies strongly indicate that the OspA antibodies elicited by the vaccines prevent human infection by neutralizing the spirochetes in the gut of the vector tick between the time of attachment and the subsequent transmission of B. burgdorferi to the host [6-8]. This unique mechanism of action suggests that development of effective neutralizing antibody levels is the critical determinant of vaccine efficacy. An additional protective effect may occur in the hosts bloodstream, although B. burgdorferi undergoes substantial antigenic change, resulting in repression of OspA expression when in contact with warm blood. The safety profile in vaccine trials that included more than 10 000 persons 15 years of age and older is highly reassuring. Participants had mild local discomfort at the injection site but did not experience serious systemic adverse reactions. Nevertheless, the FDA Advisory Committee expressed ambivalence and raised four main issues about the vaccines that require future attention. 1. Duration of protection, need for boosters, and alternative dosage schedules. Randomized clinical trials have established the efficacy of the Lyme disease vaccines at 20 months from the first dose, but longer follow-up studies are not yet available [4, 5]. Antibody levels seem to decrease rapidly, and frequent (perhaps annual) boosters will probably be necessary. Now that it is clear that a third dose is needed to achieve adequate protection levels, dosage schedules other than the current 0-, 1-, and 12-month schedules must be explored with the goal of achieving complete immunization within a single Lyme disease season. 2. Use of the vaccines in children. Children are at the highest risk for Lyme disease, but no efficacy or safety studies have been completed that will allow FDA approval of the use of Lyme disease vaccines in children. Pediatric caregivers will probably encounter substantial pressure to use the vaccines off-label. 3. Immunopathogenicity resulting in rare or late adverse reactions. Persons with a history of immune-mediated neurologic disease or arthritis, immuno-compromised persons, and pregnant women were excluded from the phase III vaccine trials. Therefore, the full safety profile is unknown, and the vaccines are not recommended for these groups. Persons with HLA-DR4 haplotype have an increased risk for chronic Lyme arthritis, a condition associated with OspA reactivity in synovial fluid. The theoretical possibility that vaccination with recombinant OspA vaccines may lead to an immunopathogenic response based on molecular mimicry led the FDA Advisory Committee to express caution about vaccinating persons who have arthritis until further studies are completed. In current studies, persons who report a history of Lyme disease do not seem to have an increased risk for serious adverse reactions, but long-term adverse events have not been evaluated [9]. 4. Effect on serodiagnosis and masking of other tick-borne diseases. Because antibodies to OspA are the basis of current enzyme-linked immunosorbent assays that are used to test for exposure to B. burgdorferi, Lyme disease vaccines will cause false-positive results. This will require the use of other diagnostic tests, such as the more expensive Western blot assay, for serologic evaluation of vaccinated patients for active Lyme disease. Western blot studies in current trials indicate that the vaccines fully protect against infection with B. burgdorferi and do not result in subclinical or modified infections that may cause late complications of Lyme disease [4, 5]. This is reassuring but requires further study, particularly in patients in whom adequate protection is not maintained through booster immunizations. Because patients with recognized Lyme disease should be carefully evaluated for concomitant babesiosis and ehrlichiosis, there is concern that patients exposed to deer ticks will not be evaluated promptly for these more serious diseases in the absence of a sentinel manifestation of Lyme disease. Humans represent a dead end for B. burgdorferi, and although vaccination will protect individual persons, it will not interrupt the epidemiologic transmission of the spirochete [10]. When a public health priority is assigned to Lyme disease vaccines, it is important to note that Lyme disease is not communicable from human to human and is associated with negligible mortality. In addition, most patients with Lyme disease (approximately 85%) develop the characteristic erythema migrans rash, which allows early recognition of disease and implementation of effective treatment with inexpensive oral antibiotics. Nevertheless, the public perception of risk for Lyme disease has increased to a level of extreme concern in areas highly endemic for this disease, and surveys indicate that demand for the vaccines will be high. The distribution of Lyme disease is highly regional. More than 90% of cases occur in 10 states (Wisconsin, Minnesota, and states on the mid-Atlantic and New England seaboards). Even in these states, the incidence of Lyme disease varies by county and by smaller geographic divisions. Although the major determinant of risk is geographic exposure, other recognized risk factors in endemic areas include outdoor work, recreational or leisure activities, and failure to take personal protective measures to avoid tick bites [10]. The uneven distribution of Lyme disease and the high degree of variation in the risk to any particular person within an endemic area confound efforts to establish uniform recommendations for vaccine use. The vaccines will not be used universally, and individual patients and health care providers (including public programs) will exercise considerable choice in their use. In regions where Lyme disease is highly endemic (incidence, >1% per year), the vaccines will probably be recommended for persons 15 to 65 years of age, excluding shut-ins and urban dwellers, who have reduced risk for tick exposure [10]. In regions of intermediate risk (incidence, 0.1% to 1% per year), recommendations will target persons whose activities increase their risk for tick exposure. The vaccines will not be recommended for persons in the regions with the lowest incidence. These stipulations leave uncertainty about whether to use the vaccine in many cases, especially in persons who travel to highly endemic areas (many of which are prime tourist destinations). In these situations, physicians will make decisions individually on the basis of duration of exposure, type of activity, and level of personal concern, just as physicians who advise persons about international travel must weigh individual factors before deciding which preventive measures to take. Perhaps a new field of domestic emporiatics (advice for persons traveling in North America) will be spawned. The excellent safety profiles of the Lyme disease vaccines and great public demand for them will make it difficult to deny vaccination to highly concerned persons. This will create problems for insurers and public vaccine programs that would like to be selective in use and reimbursement. Although the cost of the vaccines has not been determined, it is expected to be

Evidence for Vaccine Synergy between Borrelia burgdorferi Decorin Binding Protein A and Outer Surface Protein A in the Mouse Model of Lyme Borreliosis

50 to

Conformational Nature of the Borrelia burgdorferi Decorin Binding Protein A Epitopes That Elicit Protective Antibodies

100 per year; cost studies indicate a high price, especially compared with other measures (such as early recognition and treatment of Lyme disease, promotion of personal protective measures, and environmental control activities to reduce exposure to deer ticks). The need to vigorously pursue these less costly measures will not be obviated by the introduction of the vaccines. Lyme disease occurs in all age groups [10]. It is anticipated that current studies will establish the safety and immunogenicity of the Lyme disease vaccines in children and will result in the addition of these vaccines to the standard pediatric immunization schedule in areas where Lyme disease is highly endemic. Additional studies are needed in elderly persons. More user-friendly schedules (for example, 0-, 1-, and 2-month schedules) will probably be evaluated and adopted. The current Lyme disease vaccines should be considered first-generation vaccines that are useful but limited additions to our arsenal. A goal of future vaccines will be to provide longer-term protection and broader protection against variant strains of B. burgdorferi. The recent sequencing of the entire DNA genome of B. burgdorferi (>1.4 million base pairs) will undoubtedly facilitate future vaccine development [11].

Role of Lipopolysaccharide Phase Variation in Susceptibility of Haemophilus influenzae to Bactericidal Immunoglobulin M Antibodies in Rabbit Sera

ABSTRACT Mice immunized with either the predominantly vector-stage lipoprotein outer surface protein A (OspA) or the in vivo-expressed lipoprotein decorin binding protein A (DbpA) are protected againstBorrelia burgdorferi challenge. DbpA-OspA combinations protected against 100-fold-higher challenge doses than did either single-antigen vaccine and conferred significant protection against heterologous B. burgdorferi, B. garinii, andB. afzelii isolates, suggesting that there is synergy between these two immunogens.

Protective immunity elicited by recombinant bacille Calmette-Guerin (BCG) expressing outer surface protein A (OspA) lipoprotein: a candidate Lyme disease vaccine.

ABSTRACT Decorin binding protein A (DbpA) has been shown by several laboratories to be a protective antigen for the prevention of experimental Borrelia burgdorferi infection in the mouse model of Lyme borreliosis. However, different recombinant forms of the antigen having either lipidated amino termini, approximating the natural secretion and posttranslational processing, or nonprocessed cytosolic forms have elicited disparate levels of protection in the mouse model. We have now used the unique functional properties of this molecule to investigate the structural requirements needed to elicit a protective immune response. Genetic and physicochemical alterations to DbpA showed that the ability to bind to the ligand decorin is indicative of a potent immunogen but is not conclusive. By mutating the two carboxy-terminal nonconserved cysteines of DbpA from B. burgdorferi strain N40, we have determined that the stability afforded by the putative disulfide bond is essential for the generation of protective antibodies. This mutated protein was more sensitive to thermal denaturation and proteolysis, suggesting that it is in a less ordered state. Immunization with DbpA that was thermally denatured and functionally inactivated stimulated an immune response that was not protective and lacked bactericidal antibodies. Antibodies against conformationally altered forms of DbpA also failed to kill heterologous B. garinii and B. afzelii strains. Additionally, nonsecreted recombinant forms of DbpAN40were found to be inferior to secreted lipoprotein DbpAN40 in terms of functional activity and antigenic potency. These data suggest that elicitation of a bactericidal and protective immune response to DbpA requires a properly folded conformation for the production of functional antibodies.

Active and Passive Immunity against Borrelia burgdorferi Decorin Binding Protein A (DbpA) Protects against Infection

ABSTRACT The effect of phase variation of lipopolysaccharide (LPS) structure on the susceptibility of Haemophilus influenzae to complement-dependent killing by normal human sera and normal rat sera has been described previously. The phase-variable structure phosphorylcholine (ChoP) confers susceptibility to human serum, since ChoP on the bacterial cell surface binds to serum C-reactive protein and activates complement. In contrast, expression of galα1,4gal, a second phase-variable epitope that is also found on human glycoconjugates, confers resistance to human serum. We studied the role of phase variation of these structures in the susceptibilities ofH. influenzae KW20 (Rd) and a clinical isolate of nontypeable H. influenzae to killing by rabbit sera, which often possess naturally acquired complement-dependent bactericidal activity for unencapsulated H. influenzae. Expression of ChoP increased the resistance of strain KW20 to killing by bactericidal rabbit sera. In contrast, the serum resistance of a clinical isolate, H233, was unaffected by ChoP expression but was reduced by galα1,4gal expression. The rabbit sera with bactericidal activity (but not the nonbactericidal sera) all contained immunoglobulin M (IgM) antibodies able to bind to the surface of H. influenzae bacteria, as detected by flow cytometry, and contained IgM antibodies to LPS purified from strain KW20. Preincubation of sera with LPS reduced their bactericidal activity. Bactericidal activity was recovered quantitatively in an IgM-enriched fraction of sera. It is concluded that naturally occuring bactericidal activity for unencapsulatedH. influenzae is largely due to IgM antibodies directed against phase-variable structures of the LPS.