Efficacy of Leptospiral vaccine (vax-SPIRAL) against challenge

A hamster model was used to determine the protector capacity of vax-SPIRAL against the lethal infection after challenge with 10,000 LD of four strains isolated of a leptospirosis outbreaks in Nicaragua. These strains were 50 selected from a primary group of 16 belonging to the serogroups strains Ballum, Icterohemorragiae, Pomona, Serjoe and Pyrogenes. Most of these, except Pyrogenes serogroup, reproduced the infection in animal model and the most virulent representatives of each serogroup were selected for the challenge. The results of this study


Introduction
potential virulence factors (11,12).The aim of these has been to eliminate the disadvantages of Leptospirosis is a zoonosis caused by a large the use of inactivated/killed whole cell vaccines.group of pathogenic Leptospira comprises about However, any vaccine based on these 250 serovars grouped into 20 genomic species principles will be available in a short term, (1).It is a disease of worldwide distribution where although much progress has been made in the man is an accidental host (2).Contamination addressing new strategies for developing of drinking water with sewage waste is the major leptospiral vaccine safely and effective.The risk factor in cases of floods and natural disasters.inactivated whole cell vaccines have been widely That is why vaccination is recommended for used in countries such as China, Japan, Israel, people exposed to this kind of risk especially in Russia and Cuba and are currently the only those countries where natural events occur that permissible alternative to combat the scourge of lead to the development of outbreaks in certain the disease in spite of the main drawbacks seasons (3).However, despite its growing incidence reported: reactogenicity and limited protection to in humans, this practice has not been widespread serovars present in the formolution.and only a very small number of countries that In Cuba was designed, developed and apply it, mainly due to the low availability of evaluated a trivalent leptospiral vaccine adsorbed vaccines for humans and the little information on aluminum hydroxide gel, consisting of concerning to them (3, 4).inactivated whole cells of serovars indigenous At present, studies on leptospirosis vaccines strains: Canicola, Copenhageni and Mozdok.have focused on developing vaccine candidates Product registration was achieved in 1998 under based on lipopolysaccharides (5,6) lipoproteins ® the trade name of vax-SPIRAL (13), with (7,8), outer membrane proteins (9,10) and satisfactory results of safety, immunogenicity and Havana) 3-4 weeks old were used in the study.protective homologous capacity (14)(15)(16).Its use The animals had ad libitum access to a in the population has shown an overall efficiency commercial pelleted ration and drinking water. in the field of more than 60% which has The experiments were conducted according to contributed significantly to reduce morbidity and the protocol approved by IACUC (Institutional mortality caused by this disease in Cuba (17).
Animal The virulence of all hamster (Mesocricetus aureatus) that would evaluate isolates was estimated qualitatively in the Syrian the protective capacity of this vaccine against hamster model, according to the methodology strains that was circulating in that country.These described by González and colleagues (22).results were necessary to generate an approach to Groups of five animals of 45-50 g, were injected the commercial house about recommending or intraperitoneally with doses of 0.8, 0.4, 0.1 and not the formulation to Nicaraguans sanitary 0.05 mL of a bacterial suspension adjusted to 7.5 authorities to combat the outbreak.in EMJH media.The inoculated animals were Sixteen strains of clinical isolates recollected observed over a period of 14 days post infection during the period October-November 2007 from to evaluate the occurrence of clinical signs, the departments of Leon and Chinandega (after including the presence of nasal bleeding, hurricane Felix whipped Nicaragua) was used in dehydration, pilo erection and prostration.Animals this study.These strains were classified to that showed symptoms and signs of leptospirosis serogroup by the microscopic agglutination test were sacrificed following the code of ethics of (MAT) (18,19), using reference polyclonal animal protection (24) and kidneys were cultured antisera produced in the International Reference in EMJH liquid media at 28°C.The strains were Laboratory WHO (Tropical Medicine Institute, considered highly virulent if the desease occurred Netherlands).All strains were cultured in at least in all the animals inoculated with doses of Ellinghausen-McCullough-Johnson-Harris bacterial suspension 0.1 mL.If the desease of the (EMJH) (Difco Labratories) liquid media at 28 ° inoculated animals took place only at dose 0.4 mL C and were subsequently stored at -70°C in liquid the strain was considered of moderate virulence nitrogen and Fletcher's semisolid media (20,21).and for lower doses levels the strains were The strains belonging to serogroups Pomona and Icterohaemorrhagiae were faced also considered of low virulence or avirulent.Only highly virulent strains were used in the study of by MAT to the reference monoclonal antibodies L. interrogans median lethal dose (LD ).F70C7, F70C24 ( serovar 50 Copenhageni) and F46C5, F46C9, F58C2 (L.

LD determination in hamster:
The median 50 interrogans serovar Mozdok) respectively lethal dose was evaluated according to the obtained from Leptospirosis Reference methodology proposed by Reed and Muench (25) Laboratory WHO (Netherlands). in three different tests with different batches of Animals: Female Syrian hamsters (CENPALAB, animals.The animals were observed until 15 days after infection.As negative control were used were performed to generate a contingency table animals inoculated with EMJH liquid media. with the purpose of determine whether animal survival was due to vaccination and also to ® Evaluation of the vax-SPIRAL protection against compare the proportions between the different the challenge with the virulent strains: For the experimental groups.evaluation of heterologous protection (or crossprotection) of the vaccine, 50 female Results hamsters were immunized (n = 10 per group) with ® Serological classification of clinical isolates: The vax-SPIRAL .The animals received two doses sixteen clinical isolates were confirmed as of 0.5 ml, according to route, dose and schedule Leptospira spp.These strains were then classified proposed by Naranjo and colleagues (26).Fourteen to serogroup (Table-1) by MAT with polyclonal days after completing the scheme, they were serum reference.The strains belonging to serochallenged intraperitoneally with 10,000 LD 50 groups Pomona and Icterohaemorrhagiae were (0.5 ml of inoculum) of each of the highly virulent faced with specific monoclonal antibodies to strains.Fifty un-immunized animals were used as serovars Mozdok and Cophenageni and results control testing and were inoculated with a dose of obtained allowed affirm that none of the isolates challenge isolates as described above.The animals coincided serologically with the strains present in were kept under appropriate conditions and were ® the vax-SPIRAL .observed daily for a period of 14 days after which the survivors of each group were euthanised.
Virulence and LD determination: All the strains 50 The prevalence of the microorganism in the belonging to serogroups Icterohaemorrhagiae, main target organs of Leptospira infection were Pomona, Serjoe and Ballum reproduced the determined by a microbiological culture of liver infection in hamsters, with differences between and kidney in EMJH media (27).The organ them respect to the levels of lethality (Table-1).cultures were observed periodically under the The most virulent strains of each of these serogroups dark field microscope (Standard Option 25) and it were listed with the codes 3507, 6307, 8807 and was considered as a negative result the absence of 4207.In contrast none of the strains belonging to growth after 60 days of incubation at 30°C under serogroup Pyrogenes caused infection in animals, static conditions.not even after three passes in animals.LD average values of the strains 6307 and Statistical analysis: Data was analyzed at a 5% 50 8807 required inocula between 30  subsequent death of the infected animals, while inactivation provide specific protection to strains 4207 and 3507 needed an inocula below antigenically related serovars that are part of that vaccine formulation (28-30).The lack of cross the seven leptospira.These values showed, in protection among some serovars, the need for general, little variability between trials for the annual or biannual revaccination and the high same strain.In all cases, clinical signs could be frequency of side effects are serious drawbacks observed from the third until the eighth day postthat have limited the availability of these vaccines inoculation.
for human use.However, studies with vax-® Evaluation of the vax-SPIRAL protection against ® SPIRAL in Cuba show that this is safe, low the challenge with virulent strains: The results reactogenic and point out cross-protection of the active protection of hamsters immunized against serovars not included in it (12,13,16).® with vax-SPIRAL and challenged with strains of The introduction of a vaccine of this type in a high virulence showed high percentages of region must be preceded by a small study that protection.Figure-1 shows a 100% mortality in allows predicting the effectiveness of the product animals unimmunized and challenged.
to solve the health problem.This is necessary to The statistical comparison demonstrated have strains isolated from the affected region.that they do not exist significant differences These strains must be characterized by a high among the values of the survival percentages of virulence and be representative of the serotypes that ® animals immunized with vax-SPIRAL and subare in circulation (11,29,30).sequently challenged with Pomona, Icterohaemorrhagiae and Ballum.On the other hand were observed differences among these three groups regard to Serjoe.
In groups where some animals died may be noted that these deaths happened from day 10.Macroscopic analysis of the organs of the immunized animals that survived the challenge, in all cases revealed the absence of characteristic signs of infection (inflammation, hardening, bleeding or jaundice).Besides the organs cultures of in EMJH media showed the absence of microbial growth after 60 days of incubation.Contrary, the unimmunized animals where when examining the organs we showed all the hallmarks of this entity and the cultures of  II).

Traditionally it has been suggested that leptospirosis vaccines obtained by whole-cell
In the period from October to November the local health authorities in the departments of an immune response in biomodel evaluated, Leon and Chinandega obtained a group of sixteen which may be more or less successful.

Leptospira spp
The comparison of LD values for selected clinical isolates confirmed as .The 50 classification of these strains demonstrated that strains show differences between them.The lowest all were patogens and they belonged to the values were for strains belonging to serogroups serogroups Pomona (4), Icterohaemorrhagiae Icterohemorriageae and Serjoe, which showed (3), Ballum (3), Serjoe (3) and Pyrogenes (3).differences with the rest of the scores level, but all Likewise the strains belonging to the first two were below the dose known to microorganisms serogrupos mentioned previously didn't coincide belonging to these serogroups and classified as highly virulent (31).The behavior of the survival in the classification at serovar level with the ® curves of these animals was also consistent with strains present in vax-SPIRAL .
those reported by other authors (32), despite the Often the virulence of pathogens is directly differences in the LD the range of deaths was quantified in animal models by LD determination, 50 50 only five days for an average period of seven days in our case were used primarily qualitative methods of death.because although they are less accurate, are ® Vax-SPIRAL was able to protect against equally reproducible and require fewer animals.
the Leptospira strains isolated from the epidemic These methods are being more appropriate to in Nicaragua.The best results were obtained in assess the virulence of a high number of strains groups challenged with Icterohemorriageae and simultaneously (24).In this study only 13 of the Pomona that which is expected if we consider that 16 strains reproduced the infection in the animal these serogroups are included in the vaccine, model and of these only five were classified as although they do not belong to the same serovars.highly virulent.Which generally led to an early th th For the rest of the serogroups a value of survival death of animals between the 5 and 8 day post bigger than 60% was observed.These results inoculation.Coincidentally the three strains that were consistent with the conclusions of an did not reproduce the infection belonged to effectiveness clinical trials carried out in the serogroup Pyrogenes.Although there were counties Holguin and Villa Clara (Cuba) where a several passes to them in the animal model was protection of 60,4% was detected against impossible to recover.Consequently, the strains serogroups not included in the vaccine (13).It classified as high virulence were ruled to be was also recently published a study that displayed evaluated in further studies.Although this trial ® the protection of vax-SPIRAL against most was excluded strains of epidemiological studies, common strain of the outbreak in Honduras after ensures a reduction of animal suffering and costs.
hurricane Mitch (16).All these results demonstrate Also manages the negative controls immunoexplicitly heterologous cross-protection of the protection test work properly, ie values are vaccine, in the Syrian hamster model.obtained close to 100% lethality in this group that assigns greater accuracy to statistical comparisons.
Few studies have been intended to demonstrate the existence of cross-protection The loss or attenuation of virulence has been among different serogroups of Leptospira.Until a demonstrated by different investigators but its causes remain a highly contested phenomenon.In few years ago was well established that the leptospira numerous studies show substantial protection conferred after natural infection or immunization was serovar / serogroup specific differences in the expression of important antigens (33), but the concepts of immunity against this in the pathogenesis between virulent and avirulent infection have been modified in the light of isogenic strains (28).Extracellular proteins, outer current knowledge.Very recent works have membrane lipoprotein and adhesins are expressed reported experimental results that demonstrate mostly by strains virulent than their avirulent the induction of a statistically significant cross variants (29).Apparently this unequal antigenic protection as a result of immunizing leptospira architecture is reversed in uneven invasive capacity protein fractions (12,34). of these different strains, and as a result generates At the present time the theory more accepted was not possible to obtain virulent strains able to reproduce the disease in hamsters.It is important regarding the leptospira cross protection it is that to add that the eradication of this health problem outlines that cross protection exists, for those will not be possible only with the immunization related serovares that sharing a high similarity in of the affected population, this also requires the the LPS polisacaridic nucleus and for the non creation of a set of measures that include the related serovares that present key proteins vaccination of animals, decline rates of demonstrated as highly conserved factors of infestation by rodents in areas near human virulence (12).
settlements and control of animal and human Following the recommendations of the wastes; together with the training of public health WHO was demonstrated that the immunization and direction of medical personnel in order to conferred a total protection against carrier status improve epidemiological studies. in this animal model (table II).These results endorse the effectiveness of the vaccine because Acknowledgements even it is known that the carrier state in man is of Authors are thankful to the public health short duration after natural infection and usually ministry of Nicaragua for their good attention and is not a major source of leptospira spread, the the workers of the animals facilities of the human vaccines should eliminate the carrier state Research Vicepresidency, Finlay institute.in preclinical studies.Among the possible

7 x 10
cells / mL (direct counting in Petroff-Hausser Materials and Methods chamber) (Fisher Scientific) (23).This bacterial suspension came from a static culture of 5-6 days Bacterial strains and serological classification:

Figure- 1 .
Figure-1.Protection against lethal infection conferred by the ® sections of organs such as liver and kidney vaccine vax-SPIRAL in hamsters after challenge with 10,000 LD of strains 3507 (L.Icteroahemorrhagiae), 6307 (L.50 Pomona), 4207 (L.Serjoe ) and 8807 (L.Ballum) at 14 days post-inoculation.The letters on the bars represent the Discussion statistical comparison results obtained from the application of the Chi-square test with significance level p <0.05.

of virulence:
Care and Use Committee) at Finlay In October 2007, after hurricane Felix Institute to comply with biosecurity principles.whipped Nicaragua was reported a total of 6334 All work was conducted in compliance with suspected cases of leptospirosis.With the aim of regulations, policies, and principles of the Animal combating the epidemic was requested Welfare Act; the Public Health Service for Policy immunization of the affected population with on Humane Care and Use of Laboratory animals ® used in Testing, Research, and Training; the NIH vax-SPIRAL but due to the intrinsic limitations Guide for the Care and Use of Laboratory Animals. of this vaccine was necessary to conduct an experimental study in the biomodel Syrian Evaluation