Mouse Protection Test Research Articles

Back passage of haemorrhagic septicaemia (HS) vaccine seed C82 in cattle calf

Haemorrhagic septicaemia (HS) is caused by Pasteurella multocida serotypes B: 2, a Gram-negative coccobacilli bacterium. It causes fatal septicaemia with high mortality in cattle and buffaloes. Disease control and prevention are by effective HS vaccination in animals. A study was conducted on one mixed breed cattle calf for back passage of haemorrhagic septicaemia (HS) seed strain C82. Prior to bacterial inoculation in the calf, Mouse Protection Test (MPT) was conducted to ensure the calf is free from any HS antibody. The calf was injected subcutaneously with 0.5 ml dilutions of 10-3 of the bacteria incubated overnight (approximately 106 CFU/ml). After ±30 hours inoculation, post-mortem was conducted on the calf showing sign of HS. Heart blood and organ samples were collected and cultured on blood agar to determine bacteria purity. Blood and organ samples were also sent to the bacteriology laboratory in VRI for confirmatory tests. The death of the calf is confirmed by re-isolation of Pasteurella multocida Type B. Heart blood collected was processed, freeze-dried and keep as vaccine seed for further usage in vaccine production. Revival of vaccine seed is needed to ensure its pathogenicity and functionality for production purposes.

Evaluation of the Potency of the Pertussis Vaccine in Experimental Infection Model with Bordetella pertussis: Study of the Case of the Pertussis Vaccine Used in the Expanded Vaccination Program in Algeria

In Algeria, vaccination against pertussis is carried out using the whole-cell pertussis vaccine combined with the diphtheria and tetanus toxoids (DTwp). The quality control of vaccines locally produced or imported is carried out before the batch release. The aim of our work was to evaluate the potency of pertussis vaccines. In the present study, five consecutive trials of potency were conducted on samples of the same batch of (DTwp) using the mouse protection test (MPT) against experimental infection of Bordetella pertussis strain 18323, based on the Kendrick test. The virulence of B. pertussis strain 18–323 was verified by the mortality of mice, with an average LD50 of 338.92, as well as the dose of the lethal test containing a mean number of LD50 of 324.43. The (MPT) test recorded a relative potency of 8.02 IU/human dose, with 95% CL of (3.56–18.05) IU/human dose. The development of the (MPT) at the laboratory of quality control of vaccines and sera at the Pasteur Institute of Algeria was effective in evaluating the potency of whole-cell pertussis vaccines. Interestingly, our study indicates that this potency is necessary for the vaccine quality assurance. Further validation is needed to strengthen the application and routine use of the test.

Active Immunization with Pasteurella multocida Lysate Elicits Antibody that Protects Rabbits against Virulent Pasteurella multocida and Protects Mice by Passive Immunization

Background: Pasteurellosis is an economically important disease of livestock worldwide and vaccination is effective mean to control the disease outbreaks. One of alternative method for vaccine preparation could be the use of lytic phages as bacterial inactivating agents and employing “phage lysate immunogen” as an immunizing agent. Therefore, the study was undertaken to explore the protective immune response of novel phage lysate vaccine against Pasteurella multocida. Methods: A phage lysate vaccine of Pasteurella multocida was prepared using isolated lytic phage and subjected to vaccine response study in rabbits. In this experiment, two mouse fixed-dose of lysate was used and the prophylactic efficacies of lysates were evaluated in rabbits by passive mouse protection test (PMPT), western blotting and direct virulent challenge.Result: Passive transfer of antibodies to mice using post-immunization sera of rabbits protected the animals against the challenge with A: 1 strain (75%) and B: 2 strain (50%). In western blotting, a total number of 4 bands were observed in the region between 130 kDa to 25 kDa in the protein of the phage lysate. The study suggested the significance increase in humoral immunity by phage lysate in murine Pasteurellosis.

THE PROTECTIVE VALUE OF AN AUTOGENOUS BACTERIN AND TOXOID AGAINST EXPERIMENTAL CORYNEBACTERIUM PYOGENES INFECTION IN MICE

Bacteria and toxin from a borth culture of a porcine strain of Corynebacterium pyogenes were lethal to white Swiss mice within 24th of inoculation. The pyogenic factor was shown to be a component of the bacterial cells and not of the toxin. Mouse protection tests using a formolised bacterin and formolized toxoid conferred protection to experimental challenge. It is suggested that this toxoid may be of value in protecting animals against the natural infection.

PREPARATION AND COMPARATIVE EVALUATION OF HAEMORRHAGIC SEPTICAEMIA VACCINES USING EOLANE-150 AND EOLANE-170 AS OIL ADJUVANTS FOR CATTLE AND BUFFALO

Haemorrhagic septicaemia (HS) caused by Pasteurella multocida serotype B:2 is an economically important disease of cattle and buffalo, causes heavy economic losses due to sudden death of animals in developing countries like Pakistan. In this country, animals are vaccinated by alum (adjuvant) precipitated vaccine twice a year. Immunity induced through this prophylactic measure lasts for 3-4 months only. Two new HS oil based vaccines were prepared by using two new oil adjuvants such as Eolane-150 and Eolane-170. The ratio of bacterial antigen and oil adjuvants was 1:1 while bacterial dry weight was adjusted to 2 mg/ml. The addition of enrichments and aeration resulted in dense bacterial growth of Pasteurella multocida. Both new vaccines passed sterility, safety and potency tests as per OIE, 2017. Active and passive Mouse Protection Tests were performed to evaluate its potency. Indirect Haem-Agglutination (IHA) test was conducted on serum samples of two rabbits, groups each of which was vaccinated with HS oil based vaccines adjuvanted with Eolane-150 and Eolane-170. IHA indicated that immune response was higher (GMT=32) initially on 45th day to 75th day post vaccination and then declined (GMT=16) in the rabbits vaccinated with HS vaccine adjuvanted with Eolane-150, while protective immune response remained constant (GMT=16) up to ninety days post vaccination in the rabbits vaccinated with HS vaccine adjuvanted with Eolane-170. Vaccines were easy to inject with no side effects, including swelling at the injection site and longer protection as well. That would hopefully motivate the livestock owners and farmers to use this new product to protect their animals against this fatal HS disease.

Immunogenicity of Non-Living Anthrax Vaccine Candidates in Cattle and Protective Efficacy of Immune Sera in A/J Mouse Model Compared to the Sterne Live Spore Vaccine.

The Sterne live spore vaccine (SLSV, Bacillus anthracis strain 34F2) is the veterinary vaccine of choice against anthrax though contra-indicated for use with antimicrobials. However, the use of non-living anthrax vaccine (NLAV) candidates can overcome the SLSV limitation. In this study, cattle were vaccinated with either of the NLAV (purified recombinant PA (PrPA) or crude rPA (CrPA) and formaldehyde-inactivated spores (FIS of B. anthracis strain 34F2) and emulsigen-D®/alhydrogel® adjuvants) or SLSV. The immunogenicity of the NLAV and SLSV was assessed and the protective efficacies evaluated using a passive immunization mouse model. Polyclonal IgG (including the IgG1 subset) and IgM responses increased significantly across all vaccination groups after the first vaccination. Individual IgG subsets titres peaked significantly with all vaccines used after the second vaccination at week 5 and remained significant at week 12 when compared to week 0. The toxin neutralization (TNA) titres of the NLAV vaccinated cattle groups showed similar trends to those observed with the ELISA titres, except that the former were lower, but still significant, when compared to week 0. The opsonophagocytic assay indicated good antibody opsonizing responses with 75% (PrPA+FIS), 66% (CrPA+FIS) and 80% (SLSV) phagocytosis following spores opsonization. In the passive protection test, A/J mice transfused with purified IgG from cattle vaccinated with PrPA+FIS+Emulsigen-D®/Alhydrogel® and SLSV had 73% and 75% protection from challenge with B. anthracis strain 34F2 spores, respectively, whereas IgG from cattle vaccinated with CrPA+FIS+Emulsigen-D®/Alhydrogel® offered insignificant protection of 20%. There was no difference in protective immune response in cattle vaccinated twice with either the PrPA+FIS or SLSV. Moreover, PrPA+FIS did not show any residual side effects in vaccinated cattle. These results suggest that the immunogenicity and protective efficacy induced by the NLAV (PrPA+FIS) in the cattle and passive mouse protection test, respectively, are comparable to that induced by the standard SLSV.

Comparative Assessment of the Whole-cell Pertussis Vaccine Potency Using Serological and Intracerebral Mouse Protection Methods.

One of the most important QC tests of whole-cell pertussis vaccine (WCPV) is potency test. In this regard, mouse protection test (MPT) is the current potency method, which is associated with severe animal distress and large variability in results. The purpose of this study was to assess Pertussis Serological Potency Test (PSPT) as a serological alternative method to intracerebral challenge in MPT assay. In the current study, the potency of three experimental batches of WCPV (1, 2, and 3) and standard vaccine were compared using MPT and PSPT methods. In the MPT method, mice were immunized with tests and standard vaccines. After 2 weeks, they were intracerebrally challenged with Bordetella pertussis strain (18323). The potency was calculated via parallel line analysis based on the numbers of survivors 2 weeks after the challenge. Similar to MPT method, mice were immunized in the PSPT method and bled after 4 weeks. In the next step, sera were titrated by 18323-WCP-ELISA assay and potency values were estimated via parallel line analysis. Pearson correlation test was used to measure the strength of association between MPT and PSPT assay results. The potency values of the experimental laboratory batches 1, 2, and 3 in MPT assays were 11.14, 5.02, and 4.24 Iu/ml, whereas the obtained results of PSPT assays were 10.32, 4.11, and 3.06 Iu/ml, respectively. The correlation of MPT and PSPT results was 0.807. The findings of the present study demonstrated a significant correlation between MPT and PSPT results. The implementation of PSPT was more advantageous, compared to MPT due to its ethical approaches and less variability in results. The PSPT is a promising alternative method for intracerebral challenge. However, additional validation is needed to support the establishment of this method.

Erysipelas, the opportunistic zoonotic disease: history, epidemiology, pathology, and diagnosis—a review

Due to the recent multidisciplinary collaborations towards ‘One Health’, it was necessary to review this important zoonotic disease, erysipelas. Swine erysipelas also referred to as diamond skin disease has since become one of the most serious hazards of swine production worldwide. Erysipelothrix rhusiopathiae, the aetiology of erysipelas could affect pigs, turkeys, sheep, chickens, ducks, and emus. The most important animal reservoir of E. rhusiopathiae is the domestic swine. The organism is shed by diseased animals in faeces, urine, saliva, and nasal secretions, which can contaminate food, water, and soil. Swine erysipelas is made up of the hyperacute form, the septicaemia, and cutaneous (diamond) forms (acute), and the chronic form, characterised in pigs by joint lesions (arthritis), vegetative endocarditis, alopecia, and dermatitis. These signs are accompanied by poor fertility, increased prevalence of abortions, stillbirths, and small litter size. Reports of human cases are related to occupational exposure placing fishermen, butchers, slaughterhouse workers, veterinarians, housewives, poultry industry workers, and other agricultural based workers at higher risk. Diagnosis of erysipelas is based on clinical signs, gross lesions, response to antimicrobial therapy, and demonstration of the bacterium using bacterial culturing, mouse protection test, microscopy and mass spectrometry or demonstration of bacterial DNA in tissues from affected animals using PCR assays, immunohistochemical methods, and other serological methods. Swine erysipelas as an opportunistic zoonotic disease is on the increase hence, a synergistic effort should be garnered towards reducing the negative influence of this disease, through enhanced awareness of this disease amongst farmers, butchers, housewives, veterinarians, and drug researchers.

Active immunization with Brucella abortus S19 phage lysate elicits serum IgG that protects guinea pigs against virulent B. abortus and protects mice by passive immunization

Brucellosis is an economically important zoonosis of worldwide significance. Earlier (Jain et al., 2015) we reported methodology for generation of phage lysate preparations against Brucella abortus S19 using brucellaphage ‘ϕLd’. In this study, using a fixed dose (Two mouse PD100) of lysates, the prophylactic efficacies of both plain and alum gel adjuvanted lysates were evaluated in guinea pig by direct virulent challenge and passive mouse protection test (PMPT). Strong humoral and cell mediated immune responses in guinea pigs and protection comparable to S19 vaccine was observed with low dose (1.0 μg protein and 120 μg carbohydrate adsorbed on 0.1% aluminium gel). Passive transfer of antibodies to mice using d 90 post immunization sera of guinea pig protected the animals against challenge. The study suggested the significance of humoral immunity in murine brucellosis. Further, the methodology can be explored to produce a new class of immunotherapeutic agents against bovine brucellosis.

Vaccine-Mediated Activation of Human TLR4 Is Affected by Modulation of Culture Conditions during Whole-Cell Pertussis Vaccine Preparation.

The potency of whole-cell pertussis (wP) vaccines is still determined by an intracerebral mouse protection test. To allow development of suitable in vitro alternatives to this test, insight into relevant parameters to monitor the consistency of vaccine quality is essential. To this end, a panel of experimental wP vaccines of varying quality was prepared by sulfate-mediated suppression of the BvgASR master virulence regulatory system of Bordetella pertussis during cultivation. This system regulates the transcription of a range of virulence proteins, many of which are considered important for the induction of effective host immunity. The protein compositions and in vivo potencies of the vaccines were BvgASR dependent, with the vaccine containing the highest amount of virulence proteins having the highest in vivo potency. Here, the capacities of these vaccines to stimulate human Toll-like receptors (hTLR) 2 and 4 and the role these receptors play in wP vaccine-mediated activation of antigen-presenting cells in vitro were studied. Prolonged BvgASR suppression was associated with a decreased capacity of vaccines to activate hTLR4. In contrast, no significant differences in hTLR2 activation were observed. Similarly, vaccine-induced activation of MonoMac-6 and monocyte-derived dendritic cells was strongest with the highest potency vaccine. Blocking of TLR2 and TLR4 showed that differences in antigen-presenting cell activation could be largely attributed to vaccine-dependent variation in hTLR4 signalling. Interestingly, this BvgASR-dependent decrease in hTLR4 activation coincided with a reduction in GlcN-modified lipopolysaccharides in these vaccines. Accordingly, expression of the lgmA-C genes, required for this glucosamine modification, was significantly reduced in bacteria exposed to sulfate. Together, these findings demonstrate that the BvgASR status of bacteria during wP vaccine preparation is critical for their hTLR4 activation capacity and suggest that including such parameters to assess consistency of newly produced vaccines could bring in vitro testing of vaccine quality a step closer.

Protection against P. multocida B:2 using Recombinant 87kDa OMP Periplasmic Protein

Protection against P. multocida B: 2 using Recombinant 87kDa OMP Periplasmic Protein To clone and express Pasteurella. multocida (P. multocida) encoding 87 kDa omp gene in prokaryotic (secretory) vector and to study the immunogenicity of expressed protein.The genomic DNA was isolated from P. multocida B:2. Specific primers were designed to suite secretory prokaryotic vector system. PCR amplified gene was cloned in pGEMT-EASY vector (Promega, USA) and subcloned into pBAD/gIIIA prokaryotic expression vector. The protein was purified and Passive mouse protection test challenge was conducted in mice.

Whole-cell pertussis vaccine potency assays: the Kendrick test and alternative assays

Whole-cell pertussis vaccines are still widely used across the globe and have been shown to produce longer lasting immunity against pertussis infection than acellular pertussis vaccines. Therefore, whole-cell vaccines are likely to continue to be used for the foreseeable future. The intracerebral mouse protection test (Kendrick test) is effective for determining the potency of whole-cell pertussis vaccines and is the only test that has shown a correlation with protection in children. Here we review the Kendrick test in terms of international requirements for vaccine potency and critical technical points to be considered for a successful test including test validity, in-house references and statistical analysis. There are objections to the Kendrick test on animal welfare and technical grounds. Respiratory challenge assays, nitric oxide induction assay and serological assays have been developed and have been proposed as possible methods which might provide alternatives to the Kendrick test. These methods and their limitations are also briefly discussed. Establishment of validated in vitro correlates of protection has yet to be achieved. New technical developments, such as genome sequence and the use of gene microarrays to screen responses triggered by vaccine components may also provide leads to alternative assays to the Kendrick test by identifying biomarkers of protection.

Effect of vitamin AD3E supplementation for haemorrhagic septicaemia vaccine in laboratory mice

Haemorrhagic septicemia is a fatal disease in tropical countries and annual vaccination is carried out in endemic areas. It is a killed vaccine with mineral oil as the adjuvant. The protection level of animal against infectious agent can be improved by the maintenance of proper nutrient status and vitamin supplementation. This method is followed by many vaccines both in animal and human medicine to enhance both cell mediated and humoral immunity. The objective of this study was to evaluate the logarithmic protection values of haemorrhagic septicaemia (HS) vaccine by vitamin AD3E supplementation in laboratory mice model. Active mouse protection test was performed by Reed and Muench’s method to calculate the logarithmic protection value in HS vaccine using mice model. The logarithmic protection value of non-supplemented group was 6.33 and supplemented groups were 6.73 and 6.75; and were found to be significant in treatment. It was concluded that the logarithmic protection level of vaccine can be significantly improved by the addition of vitamin AD3E in mice and presumption may be the same with livestock in field situation.

Comparative analysis of the intracerebral mouse protection test and serological method for potency assays of pertussis component in DTP vaccine.

The aim of this study was to compare the PSPT standardized in-house as an alternative to MPT for potency assays of pertussis component. Statistical analyses have showed similar pertussis potency values when PSPT was compared to MPT. Significant correlation between the potency results obtained by in vivo and in vitro assays was also been observed. Results by PSPT have demonstrated reproducibility and accuracy for potency pertussis control and this approach has been considered promising for use at least during the steps of production.

Evidence of significant synergism between antibiotics and the antipsychotic, antimicrobial drug flupenthixol

Previously, the antipsychotic, non-antibiotic compound flupenthixol dihydrochloride (Fp) was shown to exhibit distinct in vitro antibacterial activity against Gram-positive and Gram-negative bacteria and to significantly protect Swiss albino mice challenged with a known mouse virulent salmonella. The present study was designed to ascertain whether this drug could efficiently augment the action of an antibiotic or a non-antibiotic when tested in combination. A total of 12 bacterial strains belonging to various genera were selected for this study and were sensitive to the antibiotics penicillin (Pc), ampicillin, chloramphenicol, tetracycline, streptomycin, gentamicin, erythromycin, ciprofloxacin, and to the non-antibiotics methdilazine, triflupromazine, promethazine, and Fp. Pronounced and statistically significant synergism (p < 0.01) was observed when Fp was combined with Pc following the disc diffusion assay system. With the help of the checkerboard method, the fractional inhibitory concentration (FIC) index of this pair was found to be 0.375, confirming synergism. This pair of Fp+ Pc was then subjected to in vivo experiments in mice challenged with Salmonella enterica serovar Typhimurium NCTC 74. Statistical analysis of the mouse protection test suggested that this combination was highly synergistic (p < 0.001, Chi-squared analysis). Fp also revealed augmentation of its antimicrobial property when combined with streptomycin, gentamicin, ciprofloxacin, and the non-antibiotic methdilazine. The results of this study may provide alternatives for the therapy of problematic infections such as those associated with Salmonella spp.

IMPROVEMENT OF RABBIT PASTEURELLOSIS VACCINE USING MONTANIDE ISA50

Montanide ISA50 was used as an adjuvant for preparation of inactivated rabbit pasteurellosis vaccine, and compared with the commercially produced oil inactivated rabbit pasteurellosis vaccine adjuvanted with mineral oil. Two groups of rabbits were inoculated separately by first dose of these vaccines followed by second dose after 21 days from the first dose, the mineral oil used for first group and ISA50 used for the second group as adjuvant, meanwhile, the third group was left as non-vaccinated control group. Cellular immunity was evaluated by using cell proliferation test and macrophage activity test (phagocytic percentage, phagocytic index). Humoral immune response was evaluated by using indirect haemagglutination test (IHA) and enzyme linked immunosorbent assay (ELISA). The results of the aforementioned tests proved that the two types of adjuvant induced protective titre against the diseases, but Montanide ISA50 oil adjuvant gave higher and longer duration of antibody level than the mineral oil. Challenge test and protection test in rabbit and mice gave similar results.

A note on a series of decamethylenebis[(4-substituted amino)quinaldinium] salts with potent antibacterial properties.

Abstract A number of decamethylenebis[4-(substituted amino)quinaldinium acetates] have been prepared and certain of these showed markedly increased antibacterial activity compared with the unsubstituted compound. Local therapeutic activity, assessed by mouse protection tests, reached a maximum in the C3–C6 range of the n-alkylamino-homologues, but increased activity was associated with increased toxicity.

British Pharmaceutical Conference Blackpool, 1949

Summary (a) A comparison has been made of the efficiency of various oily injections of penicillin using a mouse-protection test. (b) It is concluded that the most satisfactory preparation consists of procaine penicillin of small particle size dispersed in a stearate/arachis oil gel. (c) A theory is suggested for the mechanism by which aluminium stearate induces “slow-release.“ I am indebted to Dr. O. L. Davies for the design of the tests and the analysis of results, to Dr. A. R. Martin for supervision of the biological work, and to Mr. A. G. Fishburn for help in the preparation of the paper.

Humane endpoints in vaccine potency testing

Vaccine potency and safety testing is characterized by extensive use of laboratory animals and a relatively high percentage of test methods that involve severe pain and distress. This is particularly true for tests that are based on infection or challenge with a virulent microorganism. Traditionally, vaccine potency tests on inactivated vaccines require a vaccination–challenge procedure using severe clinical signs or even lethality as endpoints. For several of these vaccines, 3R methods have been developed that include a nonclinical endpoint, ultimately resulting in reduction of animal numbers and a significant decrease in severity level. An example is the use of serology in potency testing of tetanus and diphtheria toxoid vaccines. For some potency tests, however, replacement of the challenge procedure is not (yet) possible, and the implementation of humane endpoints might be an approach to limit the level and duration of pain and distress. The application of these endpoints is now allowed in most pharmacopoeias. Establishing humane endpoints in vaccine potency testing requires the identification of parameters that are predictive of death, or severe clinical signs, in the animal during the observation period. As a case study, we present the results of work we performed on the identification of humane endpoints in whole cell pertussis (wP) vaccine potency testing (the mouse protection test or the Kendrick test). In this potency test, mice are challenged by intracerebral route 14 days after immunization with a lethal dose of virulent B. pertussis microorganisms. Animals are observed for 14 days, and the number of mice per dose group surviving this period is used for probit analysis and estimation of potency. We have studied two types of humane endpoints: clinical signs and pathophysiological parameters (body weight and body temperature). Clinical signs in a wP potency test range from piloerection, hunched back posture, apathy, and convulsions to moribund condition. Also body temperature drops, and animals lose up to 50% of their body weight post-challenge. Parameters were “validated” for relevance (prediction of death within the observation period) and reliability. Recommendations are given for implementation of humane endpoints in vaccine potency testing, also taking into account potential obstacles.

Recombinant Diabody-Based Immunocapture Enzyme-Linked Immunosorbent Assay for Quantification of Rabies Virus Glycoprotein

The potency of rabies vaccines, determined using the NIH mouse protection test, can be directly correlated to the amount of rabies virus glycoprotein (RV GP) present in the vaccine. In an effort to develop a simple and sensitive enzyme-linked immunosorbent assay (ELISA) using recombinant diabody for quantification of RV GP, the variable heavy (V(H)) and light chain (V(L)) domains of an RV GP-specific human monoclonal antibody (MAb) secreted by a human x mouse heterohybridoma (human MAb R16E5) was amplified, linked using splicing by overlap extension PCR (SOE PCR), and expressed as a recombinant diabody (D06) in the pET28a bacterial expression system. The diabody D06 was purified by immobilized metal affinity chromatography on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The purified diabody was used in combination with a well-characterized RV GP-specific mouse MAb, M5B4, to develop an immunocapture ELISA (IC-ELISA) for the quantification of RV GP in human rabies vaccine preparations. The maximum detection limit of the IC-ELISA using the M5B4-D06 combination was up to 31.25 ng/ml of RV GP. The specificity of the diabody was established by its nonreactivity toward other human viral antigens as determined by ELISA and toward RV GP as determined by immunoblot transfer assay and competitive ELISA with the parent human MAb R16E5 and MAb M5B4. The adjusted r(2) value obtained by the regression through the origin model was 0.902, and the equation for predicted potency values for M5B4-D06-based IC-ELISA and MAb M5B4 IC-ELISA were 0.5651x and 0.8044x, respectively, where x is the estimate of RV GP from the IC-ELISA in micrograms. Analysis of variance (ANOVA) results showed the estimates of the two methods differed significantly (P < 0.001), while the predicted potencies by the two tests did not differ significantly (P > 0.05). The IC-ELISA can be readily adapted to measure the RV GP content in purified antigen, and a vaccine can be formulated based on the estimated GP.