Four-dimensional stereotactic radiotherapy for early stage non-small cell lung cancer: a comparative planning study.

In this study we sought to assess the potential of the respiratory tumor tracking system of the CyberKnife to administer 3 fractions of 15 Gy in the treatment of early stage non-small cell lung cancer (NSCLC). The CyberKnife plans were compared to those developed for 3-D conformal radiotherapy (3-D CRT) administering 20 fractions of 3 Gy based on a slow CT. Ten patients with stage I NSCLC, who were previously treated with 3-D CRT, were re-planned with the CyberKnife treatment planning system. In the 3-D CRT plan, the planning target volume (PTV) included the gross tumor volume (GTV)(slow) and a 15-mm margin, whereas in the CyberKnife plan the margin was 8 mm. The physical doses from both treatment plans were converted to normalized total doses (NTD) using the linear quadratic model with an alpha/beta(tumor) of 10 Gy and alpha/beta(organs at risk (OAR)) of 3 Gy. The average minimal and mean doses administered to the PTV with the CyberKnife and 3-D CRT were 93 and 115.8 Gy and 61 and 66 Gy, respectively (p<0.0001). The mean V(20) of the CyberKnife and 3-D CRT plans were 8.2% and 6.8%, respectively (p=0.124). Both plans complied with the OAR constraints. In conclusion, 4-dimensional stereotactic radiotherapy can increase the minimal and mean biological dose with 51% and 75%, in comparison with 3-D CRT without significantly increasing the V(20), respectively.

Loss of the common "A" determinant of hepatitis B surface antigen by a vaccine-induced escape mutant.

A previous study (Carman, W. F., A. R. Zanetti, P. Karayiannis, J. A. Waters, G. Manzillo, E. Tanzi, A. J. Zuckerman, and H. C. Thomas. 1990. Lancet. 336:325-329) demonstrated a variant hepatitis B surface antigen (HBsAg) in a vaccinated child born to a hepatitis B virus-infected mother. A substitution of arginine for glycine at amino acid 145 in HBsAg was observed. In this study the effect of this substitution on the common "a" determinant of this protein, against which protective immunity is directed, is investigated. Using recombinant HBsAg with and without the amino acid substitution, the binding of monoclonal antibodies that recognize different epitopes of the "a" determinant, was shown to be destroyed by the presence of arginine at amino acid 145. In convalescent and vaccinee sera, antibody binding to HBsAg was not inhibited by the variant HBsAg. Immunization with the variant HBsAg, although eliciting a high titer antibody that recognized the variant, produced a low titer of antibody recognizing the native protein. Studies in mice demonstrate that the immunogenicity of the variant protein is also substantially altered. The data presented here demonstrate that this variant evades the known protective anti-HBs response and lends support to the suggestion that this mutation arose as the result of immune pressure.

Simultaneous synthesis and assembly of various hepatitis B surface proteins in Saccharomyces cerevisiae.

Yeast transposon of class-1-based vectors, allowing integration at a series of chromosomal loci by homologous recombination with resident transposons, were constructed. Using such vectors, we have introduced several copies of an expression cassette encoding the major hepatitis B surface protein as well as expression cassettes encoding the middle (M) or/and the large (L) surface protein into Saccharomyces cerevisiae. In extracts of such strains, coassembly of the different proteins into a single lipoprotein structure is observed. This was demonstrated by immunoprecipitation of the major protein using monoclonal antibodies directed specifically against epitopes that are present only on the M or the L protein. These results show that hepatitis B surface antigen envelope proteins synthesized in yeast are able to assemble into structures composed of different polypeptides. This opens the possibility of producing in yeast a variety of particles carrying well-defined amounts of preS epitopes on their surface. Also, one can envisage the production of mixed particles containing different foreign epitopes on their surface, in defined relative abundance, which could be useful for vaccine applications.

Immunization of mice by recombinant OspA preparations and protection against Borrelia burgdorferi infection induced by Ixodes ricinus tick bites.

The wide distribution of Borrelia burgdorferi, the spirochete causing Lyme borreliosis, represents a human health hazard in many areas of the world. Vaccination has been proposed as an effective prevention strategy. Vaccination experiments were conducted with preparations of recombinant outer surface protein A (OspA) derived from Borrelia burgdorferi strain ZS7. Mice received three doses (1 microgram each) of the antigens adsorbed to aluminum hydroxide. A strong immune response to the vaccine antigen was observed. Mice were challenged after immunization, using Ixodes ricinus nymphal ticks infected with Borrelia burgdorferi strain ZS7. Infection was investigated by ear biopsy culture, xenodiagnosis with uninfected larvae and serological response to Borrelia burgdorferi antigens. All unimmunized control animals were found to be infected, while all immunized animals were found to be protected against infection by Borrelia burgdorferi. In addition, most adult ticks derived from nymphs that fed on immunized mice were found to be free of spirochetes.

Dosimetric consequences of tumor mobility in radiotherapy of stage I non-small cell lung cancer--an analysis of data generated using 'slow' CT scans.

BACKGROUND: The target coverage for radiotherapy of early-stage lung cancer was evaluated using two different CT techniques. MATERIALS AND METHODS: A conventional planning CT scan and two limited scans of the tumor region were performed in seven patients with peripheral tumors. Three 'slow' scans (slice thickness 4mm, index 3mm, revolution time 4s/slice) were then performed, followed by three-dimensional image registration. Planning target volumes (PTV) were generated using these GTV-PTV margins: (a) 1cm (PTV1.0); (b) 1.5 cm (PTV1.5); and (c) 0.9, 1.0, and 0.9 cm ('PTV(clinical)') when set-up errors are avoided. RESULTS: PTVs derived from three 'slow' scans missed 1.9% of the volume derived from three planning scans for an immobile tumor and 9.3% in the case of a mobile tumor. For an immobile tumor, PTV1.5 achieved comparable coverage to that achieved using PTVclinical, which was generated from three 'slow' scans and a planning scan. For a mobile tumor, PTV(1.5) covered only 89% of the volume captured by PTVclinical. PTV1.0 resulted in inadequate target coverage in all the patients. Reductions in potential lung toxicity (V20) were achievable in six patients despite the larger GTVclinical when treatment set-up errors were minimized. CONCLUSIONS: PTVs derived using 'slow' CT scans consistently produce superior target coverage than that using conventional scans. This may account for the poor local control observed in stage I lung cancer.

An evaluation of two techniques for beam intensity modulation in patients irradiated for stage III non-small cell lung cancer.

In locally advanced lung cancer, the use of high dose radiotherapy (RT) and/or concurrent chemo-RT is associated with significant pulmonary and esophageal toxicity. Despite a 3D conformal RT technique and the omission of elective mediastinal fields, three (of ten) patients with inoperable stage 3 NSCLC who were treated with induction chemotherapy (carboplatin-paclitaxel) followed by RT to 70 Gy, developed symptomatic radiation pneumonitis. In this planning study, the actual treatment plans of all ten patients were compared to plans derived using two beam intensity-modulated (BIM) techniques, for which similar geometrical beam setup parameters were used. In the first technique (BF-BIM), cranial and caudal boost fields were applied in order to allow field length reduction. The second technique (C-BIM) utilised 3-D missing-tissue compensators for all radiation beams. Both BIM techniques resulted in a significant sparing of critical normal tissues and the C-BIM technique was superior in all cases. When compared to the actual RT technique used for treatment, a reduction of 8.1+/-4.7% (1 S.D.) was observed in the mean lung dose for the BF-BIM plan, vs. 20.3+/-5.8% (1 S.D.) for the C-BIM plan. Similar reductions were observed in the percentage of the total lung volume exceeding 20 Gy (V(20)) for these techniques. BIM techniques appear to be a promising tool for enabling radiation dose-escalation and/or intensive concurrent chemo-RT in inoperable lung cancer.

Animal models for meningococcal disease.

There are many in vitro systems for the study of meningococcal pathogenesis, but it is only in animal models of infection that the interactions of the bacteria with whole tissues and the humoral and cellular immune systems can be assessed. Animal-infection models are also of great importance for the assessment of the protective efficacy of existing and candidate vaccines. However, the relevance of these animal models to human disease and how well protection assessed in them corresponds to protection against human disease, must always be considered. Animal models for pathogenic Neisseria have been previously reviewed (1).

SU-E-T-647: Plan Quality in Computerized Non-Coplanar IMRT Beam Angle Optimization is Highly Dependent on the Extent of the Beam direction Search Space.

PURPOSE: To investigate the relationship between plan quality and the extent of the beam direction search space in computerized beam angle selection for generating optimal (non-coplanar) IMRT plans for prostate SBRT with dose distributions simulating HDR brachytherapy. METHODS: iCycle (1) was used to investigate the relationship between plan quality and the extent of the set of beam directions available for plan generation. For a group of 10 prostate patients, optimal plans were generated for 5 direction search spaces. For coplanar treatments (CP set), 72 orientations were available for selection (separation 5°). The fully non-coplanar set (F-NCP) included the CP directions plus 430 directions spread over the sphere. The CK set contained the directions available at the robotic Cyberknife unit. CK+ and CK++ were extensions of CK to investigate some of its characteristics. Generated plans were in accordance with our clinical SBRT protocol for Cyberknife treatment, delivering 4 fractions of 9.5 Gy. Adequate PTV coverage had the highest priority. Reduction of rectum dose was the highest OAR priority. RESULTS: The mean PTV coverage (V95) of all SBRT plans was 99% ï,± 0.9% (1 SD). F-NCP plans had most favorable OAR dose parameters, while for coplanar plans OAR doses were highest. Compared to coplanar treatment, rectum Dmean/V60 were 25% / 37% and 19% / 21% lower in F-NCP and CK plans. Higher rectum dose for the Cyberknife set compared to F-NCP was not caused by a lack of posterior beams for Cyberknife. For all search spaces, reduction in OAR dose only leveled off with > 20 beams in the plans (for CP, rectum V60 in 25 beam plans was reduced by 64% compared to 11 beams). In the non-coplanar set-ups, there was a preference for beams with a (large) lateral component. CONCLUSIONS: Plan quality clearly improved with the extent of the beam direction search space (coplanar worst), and the number of beam directions in the plan (25 clearly better than 11).(1) Breedveld S, Storchi P, Voet P, Heijmen B, Med Phys 2012; DOI: 10.1118/1.3676689.

SU-E-T-617: Towards Fully Automated Multi-Criterial Plan Generation: A Prospective Clinical Study.

PURPOSE: To prospectively compare plans generated with iCycle, an in-house developed algorithm for fully automated multi-criterial IMRT beam profile and beam orientation optimization (Breedveld, Med. Phys. 2012), and plans manually generated by dosimetrists with the clinical treatment planning system. METHODS: For 20 randomly selected head-and-neck cancer patients with various tumour locations (of whom 13 received sequential boost treatments) we offered the treating physician the choice between an automatically generated iCycle plan and a manually optimized plan following standard clinical procedures. While iCycle used a fixed'wish-list' with hard constraints and prioritised objectives, the dosimetrists manually selected the beam configuration and fine-tuned the constraints and objectives for each IMRT plan. Dosimetrists and treating physicians were not informed in advance whether a competing iCycle plan was made or not. The two plans were simultaneously presented to the physician who then selected the plan to be used for treatment. For the patient group, we quantified differences in PTV coverage and sparing of critical tissues. RESULTS: In 32/33 plan comparisons the physician selected the iCycle plan for treatment. This highly consistent preference for automatically generated plans was mainly caused by improved sparing for the large majority of critical structures. With iCycle, the NTCPs for parotid and submandibular glands were reduced by 2.4% ± 4.9% (maximum: 18.5%, p=0.001) and 6.5% ± 8.3% (maximum: 27%, p=0.005), respectively. The reduction in mean oral cavity dose was 2.8 Gy ± 2.8 Gy (maximum: 8.1 Gy, p=0.005). For swallowing muscles, esophagus and larynx, the mean dose reduction was 3.3 Gy ± 1.1Gy (maximum: 9.2 Gy, p<0.001). Moreover, for 15 patients, the target coverage was improved as well. CONCLUSIONS: In 97% of cases, the automatically generated plan was selected for treatment because of superior quality. Apart from improved plan quality, automatic plan generation is economically attractive because of reduced workload.

HepG2 cell binding activities of different hepatitis B virus isolates: inhibitory effect of anti-HBs and anti-preS1(21-47).

The antigenic relationships among different hepatitis B virus (HBV) isolates were investigated by using monoclonal antibodies (MAbs) specific for HBs, preS2 (pHSA binding site), and preS1 (hepatocyte receptor-binding site) epitopes in a double immunoradiometric assay. In order to define possible functional differences resulting from structural and antigenic differences in the HBV env protein, the HBV isolates were compared in an in vitro cell-binding assay based on the attachment of 125I-labeled HBV to human hepatoma HepG2 cells. We provided evidence for a variability of the expression of preS1 and preS2 specificities in the peplomer (glyco)protein of HBV depending on dly subtype of HBsAg, which could affect the viral infectivity. We showed that the integrity of the HBV envelope structure associated with a large expression of preS1(21-47) epitopes is an essential factor for effective binding to HepG2 cells. Interestingly, the HBs-specific MAbs directed to disulfide-bond-dependent epitopes were found to be the best inhibitors of the preS1-HepG2 cell interaction (greater than 50%, at the final concentration of 0.5 micrograms/ml). The MAb F35.25 directed to the preS1(21-47) sequence corresponding to the hepatocyte receptor recognition site was, however, also found to inhibit binding. Thus, our results demonstrate the abilities of both anti-HBs and anti-preS(21-41) to block the attachment of complete HBV particles to HepG2 cells, suggesting that these antibodies should be virus neutralizing and would be expected to confer protection against reinfection.

Correlation between in vivo humoral and in vitro cellular immune responses following immunization with hepatitis B surface antigen (HBsAg) vaccines.

To study the regulation of the human immune response to hepatitis B surface antigen (HBsAg) we have carefully monitored the in vivo humoral and in vitro cellular immune responses to HBsAg in 50 subjects receiving four doses of hepatitis B vaccine according to a 0, 1, 2, 12 month vaccination scheme. Twenty-three subjects were given a plasma-derived vaccine (Hevac B) and 27 received a recombinant HBsAg vaccine (yeast-derived; Engerix-B). The humoral and cellular immune responses were measured before vaccination (day 0); 6 days after the second dose (day 36); 6 days (day 66), 2 months (day 120) and 10 months (day 365) after the third dose and 1 month after the fourth dose (day 395). Based on the kinetics of the humoral immune responses, the vaccinees could be classified into fast, intermediate and slow/non-responders. Based on the magnitude of the immune response (anti-HBs titre) on day 395, the vaccinees could be divided into high (> or = 2000 U l-1) and low (< or = 2000 U l-1) responders. A close correlation between the kinetics and the magnitude of the humoral immune response was observed. The in vivo anti-HBs response was measured using commercially available immunoradiometric assays. The in vitro cellular immune response was measured using an HBsAg-specific lymphoproliferation assay. Because of interassay variability the results were considered as dichotomous variables (proliferation versus non-proliferation) for further data analysis. A statistically significant correlation was observed between the kinetics and magnitude of the humoral immune response on the one hand and the in vitro anti-HBs response on the other hand.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunization with a polyvalent OspA vaccine protects mice against Ixodes ricinus tick bites infected by Borrelia burgdorferi ss, Borrelia garinii and Borrelia afzelii.

Sequence variability of the outer surface protein (Osp) A among Borrelia burgdorferi sl species suggests that a monovalent OspA vaccine may not protect against the various Borrelia present in Eurasia. Here, we confirmed that a monovalent recombinant OspA (rOspA) vaccine does not protect mice against Ixodes ricinus mediated infection with B. burgdorferi ss, Borrelia garinii and Borrelia afzelii. However, when mice were vaccinated with a cocktail of various rOspA from these three species, they were protected, and all challenge ticks that fed on them were cleared of their spirochetes. These results showed that a multiple OspA antigens vaccine, compatible with human use, was very efficient at protecting mice against B. burgdorferi ss, B. garinii, and B. afzelii.

Vaccination of natural reservoir hosts with recombinant lipidated OspA induces a transmission-blocking immunity against Lyme disease spirochaetes associated with high levels of LA-2 equivalent antibodies.

As observed in humans, immune responses in naturally infected reservoir hosts of Borrelia burgdorferi sensu lato rarely target the outer surface proteins (Osp) A and B of Lyme disease spirochaetes. The absence of protective immunity in such hosts following tick-borne infection allows them to play an effective role in the maintenance of Lyme borreliosis in nature. Therefore, the question was addressed whether one of the most prominent natural reservoir host species of B. burgdorferi s.l. in Europe, the yellow-necked mouse (Apodemus flavicollis), may lack the ability to elicit transmission-blocking antibodies to Lyme borreliosis spirochaetes. Yellow-necked mice were immunized with a recombinant lipidated OspA from B. burgdorferi sensu stricto or with high numbers of UV-irradiated whole spirochaetes. All immunized mice, but not untreated controls, developed polyclonal humoral immune responses to OspA (31 kDa). Serum antibodies of animals vaccinated with the recombinant OspA contained high levels of antibody to an epitope of OspA, defined by the monoclonal antibody LA-2, whereas only low levels of LA-2 equivalent antibodies could be detected in sera from animals immunized with killed spirochaetes. Ixodes ricinus ticks infected with B. burgdorferi s.s. lost their spirochaete load after feeding on animals with high levels of LA-2 equivalent antibody; ticks feeding on animals which had only low or undetectable serum levels of LA-2 equivalent antibodies retained their spirochaete infection. Furthermore, animals with high levels of LA-2 equivalent antibody were protected against spirochaete infection. Our study shows that natural mouse reservoir hosts are highly competent to generate transmission-blocking antibodies after vaccination with a lipidated recombinant OspA and indicates that antibodies to the LA-2 epitope play a key role in the destruction of B. burgdorferi s.s. within feeding Ixodes ricinus ticks.

Clinical and immunological assessment of a candidate Lyme disease vaccine in healthy adults: antibody persistence and effect of a booster dose at month 12.

A candidate Lyme vaccine was administered to 20 adult volunteers following a 0, 1, 2 months vaccination schedule, with a booster at 12 months. An immune response, assessed as 'LA-2 equivalent' antibody titres using an inhibition ELISA, was induced in all vaccinees which persisted until the booster. Titres were increased 25-fold following the booster and persisted through month 24. There was a good correlation between 'LA-2 equivalent' antibody titres and a bactericidal assay (r2 = 0.86). Local symptoms were mild, resolving spontaneously within 72 h, with no reports of rash, arthralgia or other systemic symptoms. This Lyme vaccine was safe, well-tolerated and elicited an antibody response in all volunteers which persisted at least 12 months after the booster.

Immunological properties of recombinant HBsAg produced in yeast.

Although currently available plasma-derived vaccines (PDV) against hepatitis B based on the surface antigen of the virus (HBsAg) are well-tolerated and effective, their supply is limited and time-consuming controls are necessary to assess their safety. It is therefore desirable that an alternative source of HBsAg be found. Recombinant DNA technology has provided the possibility of obtaining HBsAg in large quantities. However, it is important that a recombinant DNA hepatitis B vaccine be not only antigenically similar but also elicits a similar immune response in humans. Using the Ausria kit, the recombinant DNA vaccine of SmithKline Biologicals produced in yeast appears to have a higher antigenic content than a reference plasma-derived HBsAg preparation of similar purity when compared at equivalent protein concentrations. In competition experiments, however, antibodies obtained by immunization with PDV similarly recognized yeast-derived and plasma-derived antigens. Monoclonal antibodies directed to the common a determinant of the whole virus were also used to identify distinct epitopes on the recombinant DNA vaccine. The yeast-derived HBsAg is therefore antigenically similar to plasma-derived HBsAg. The yeast-derived vaccine (YDV) was highly immunogenic in mice, rabbits, goats, monkeys, chimpanzees, and humans. High titres of anti-HBs were reached in humans after three doses administered at 0, 1, and 6 months. The antibodies raised in humans after three doses of YDV were predominantly directed to the common a determinant. Competition studies using monoclonal antibodies raised against the whole virus showed that the antibodies had the same specificity as the antibodies induced by PDV. The affinity for the plasma-derived antigen of antibodies stimulated by YDV and PDV and antibodies present in the sera of convalescent subjects were also similar. Finally, competition experiments showed that the antibodies induced in humans by YDV and antibodies from convalescent subjects were directed to the same binding sites of the plasma-derived antigen. These studies indicate that the yeast-derived vaccine is immunologically similar to the plasma-derived vaccines both in vitro and in vivo and can therefore be expected to have similar protective efficacy.

Inhibition of Borrelia burgdorferi-tick interactions in vivo by outer surface protein A antibody.

Borrelia burgdorferi outer surface protein (Osp) A is preferentially expressed by spirochetes in the Ixodes scapularis gut and facilitates pathogen-vector adherence in vitro. Here we examined B. burgdorferi-tick interactions in vivo by using Abs directed against OspA from each of the three major B. burgdorferi sensu lato genospecies: B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii. Abs directed against B. burgdorferi sensu stricto (isolate N40) destroy the spirochete and can protect mice from infection. In contrast, antisera raised against OspA from B. afzelii (isolate ACA-1) and B. garinii (isolate ZQ-1) bind to B. burgdorferi N40 but are not borreliacidal against the N40 isolate. Our present studies assess whether these selected OspA Abs interfere with B. burgdorferi-tick attachment in a murine model of Lyme disease with I. scapularis. We examined engorged ticks that had fed on B. burgdorferi N40-infected scid mice previously treated with OspA (N40, ACA-1, ZQ-1, or mAb C3.78) or control Abs. OspA-N40 antisera or mAb C3.78 destroyed B. burgdorferi N40 within the engorged ticks. In contrast, treatment of mice with OspA-ACA-1 and OspA-ZQ-1 antisera did not kill B. burgdorferi N40 within the ticks but did effectively interfere with B. burgdorferi-I. scapularis adherence, thereby preventing efficient colonization of the vector. These studies show that nonborreliacidal OspA Abs can inhibit B. burgdorferi attachment to the tick gut, highlighting the importance of OspA in spirochete-arthropod interactions in vivo.

Plasmodium falciparum: recombinant baculoviruses direct the expression of circumsporozoite proteins in Spodoptera frugiperda cell cultures.

The DNA coding for the circumsporozoite protein (CPS) of Plasmodium falciparum has been cloned into the baculovirus expression vector pAcYM1 and expressed in Spodoptera frugiperda (Sf9) insect cells. Three DNA constructs have been made: the first one directs the synthesis of the complete CSP (aa 1-412), the second leads to the production of a species devoid of the anchor domain (aa 1-391) and the third one to a molecule lacking both signal and membrane anchor sequences (aa 18-391). All three recombinant CPS were produced at about 3 micrograms per 10(6) infected cells and were characterized in terms of immunoreactivity and apparent molecular weight. Analytical purification of the recombinant proteins was achieved by a combination of heat treatment, acidification, isoelectric focusing and ion exchange chromatography. The purified material, when injected into mice, generated only modest antibody responses, although antisera from immunized mice reacted with control CSP antigens carrying or not the major immunodominant repeat region.

Reactivity with a specific epitope of outer surface protein A predicts protection from infection with the Lyme disease spirochete, Borrelia burgdorferi.

The response to recombinant vaccines for Lyme disease was studied to determine serum antibody levels effective in protecting against tick-transmitted infection. Data presented here demonstrate a significant correlation between antibody to an epitope on outer surface protein A (OspA) and protection against infection with Borrelia burgdorferi in canines and mice. A competitive enzyme-linked immunosorbent assay was developed to measure antibody to a site on OspA, defined by monoclonal antibody LA-2. Comparison of LA-2 titers against infection of canines and mice following vaccination and challenge established a predicted value for LA-2 titers. The statistical relationship between serum antibody levels and protection was calculated by logistic regression analysis. The statistical model predicted that an LA-2 titer of 0.32 microg equivalents (eq) per ml correlated to an 80% predicted probability of protection for both mice and dogs. This value was used to classify mice and dogs as to their protected status at the time of tick exposure. The LA-2 cutoff titer (0.32 microg eq/ml) correctly classified all dogs (n = 13) and mice (n = 44) that failed to become infected. By contrast, 20 of 22 dogs and 28 of 31 mice with titers of less than 0.32 microg eq/ml became infected. On the basis of these results, we conclude that an LA-2 titer is a reliable indicator of immune status for estimating immune protection following use of OspA-based vaccines for B. burgdorferi sensu stricto.

Evaluation of the safety, reactogenicity and immunogenicity of three recombinant outer surface protein (OspA) lyme vaccines in healthy adults.

The safety, reactogenicity and immunogenicity of three candidate Lyme vaccines based on recombinant outer surface protein (OspA) presented in either lipidated or unlipidated forms, were assessed in 300 seronegative volunteers. Subjects received three doses of one of the three formulations at monthly intervals and were evaluated for antibody levels and the presence of symptoms after each dose. All formulations proved to be safe, the majority of local reactions being reported as mild, and all general symptoms were perceived to be either-mild or moderate in intensity. No subject refused a subsequent vaccine dose. All subjects were tested for both anti-OspA IgG and LA-2 equivalent antibodies up until day 84. All three vaccines induced an immune response but subjects who received lipoprotein OspA had the highest anti-OspA IgG and LA-2 equivalent GMTs after each dose and this was also true for the subset of subjects tested on day 180. The lipoprotein OspA group also had the largest number of subjects who remained seropositive for anti-OspA IgG antibodies. As the lipoprotein formulation produced the strongest immune response, with symptoms which were acceptable to all the vaccinees, we suggest further development of this vaccine.

Antigenic conservation of an immunodominant invariable region of the VlsE lipoprotein among European pathogenic genospecies of Borrelia burgdorferi SL.

Lyme disease is caused by genetically divergent spirochetes, including 3 pathogenic genospecies: Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii. Serodiagnosis is complicated by this genetic diversity. A synthetic peptide (C(6)), based on the 26-mer invariable region (IR(6)) of the variable surface antigen of B. burgdorferi (VlsE), was used as ELISA antigen, to test serum samples collected from mice experimentally infected with the 3 genospecies and from European patients with Lyme disease. Regardless of the infecting strains, mice produced a strong antibody response to C(6), which indicates that IR(6) is antigenically conserved among the pathogenic genospecies. Twenty of 23 patients with culture-confirmed erythema migrans had a detectable antibody response to C(6). A sensitivity of 95.2% was achieved, with serum samples collected from patients with well-defined acrodermatitis chronica atrophicans. Fourteen of 20 patients with symptoms of late Lyme disease also had a positive anti-IR(6) ELISA. Thus, it is possible that C(6) may be used to serodiagnose Lyme disease universally.