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Autogenous vaccine field trial for epizootic hemorrhagic disease virus and bluetongue virus does not result in high titer to homologous virus serotypes
Abstracts / International Journal of Infectious Diseases 53S (2016) 4–163
20.173 Epitiope based DNA vaccine design using epitopes predicted from Zika virus polyprotein P.S. Slathia ∗ , P. Sharma Shri Mata Vaishno Devi University, Biotechnology, Jammu, J&K/IN Purpose: Recent outbreaks of Zika virus have made the need for developing a vaccine imperative. The polyprotein sequences of the virus have been used for finding epitopes and designing an epitope based DNA vaccine using bioinformatics tools has been attempted in this study. Methods & Materials: NCBI Protein database was used as a source of polyprotein sequences of Zika virus. The sequences were studied for conservancy by MegAlign tool of DNASTAR Lasergene 8.0. NetMHCPan, NetMHCIIPan and IEDB analysis resource tools were used for finding Class I MHC binding peptides, Class II MHC binding peptides and B cell epitopes respectively. From the pool of binding peptides for MHC I CTL epitopes were predicted by CTLPred and NetCTL servers. The selected epitopic peptides were analyzed for similarity to human protein by BLASTp tool. AlgPred server was used for finding out the presence of allergenic peptides. The peptides were joined in tandem differently for MHC I, MHC II and B cell epitopes to generate three peptide sequences. The peptide properties were predicted by ProtParam of ExPasy tools. These peptides were reverse translated using Human Codon Usage table by Sequence Manipulation Suite. These gene sequences were then separately cloned into pVAC vector and the vector map was generated PlasMapper server. Results: 114 full length sequences of Zika virus polyprotein were retrieved; partial sequences were omitted for the study. The alignments revealed high degree of conservancy among the sequences and therefore consensus sequence was used for predictions. The MHC I & II peptides that were selected had no similarity to human protein sequences, were represented only once in the final sequence, were the strongest binders for the respective allele and had no allergenic properties. For the B cell epitopes only sequential or linear epitopes were predicted and the epitopes in the final sequence had all the properties as mentioned for MHC I & II except for allelic specificity. Cloning using human codons ensures the expression in humans. Conclusion: The DNA vaccine vector generated in the study has been rationally designed using existing principles of epitope based vaccine design and could be used after lab validation studies. http://dx.doi.org/10.1016/j.ijid.2016.11.364 20.174 Evaluation of monoclonal antibodies against pfMSP10 for the prospective use on in vitro growth inhibition assays of peruvian P. falciparum isolates E. Villasis a,∗ , J. Bendezu a , K. Garro a , O. Nolazco a , K. Torres b , B. Infante a , D. Gamboa b , J. Vinetz c a
UNIVERSIDAD PERUANA CAYETANO HEREDIA, CIENCIAS CELULARES Y MOLECULARES, Lima/PE b UNIVERSIDAD PERUANA CAYETANO HEREDIA, LIMA, Lima/PE c University of California, San Diego, San Diego, CA/US Purpose: The aim of this study is to evaluate monoclonal antibodies (mAb) generated against pfMSP10 (Merozoite surface
149
protein 10) and their functional role to inhibit the invasion of Peruvian P. falciparum isolates into red blood cells (RBC) by using the Growth inhibition Assay (GIA) in vitro Methods & Materials: Seven mAb and one polyclonal antibody were synthetized and evaluated by Western Blot (WB) against the recombinant MSP10 protein (rMSP10). Synchronized and purified schizonts from P. falciparum 3D7 and their concentrated supernatant were obtained by ultrafiltration. Detection of pfMSP10 protein was also evaluated in synchronized ring stage of P. falciparum cultures from 1 to 12 hours post-invasion. IFA assays were also carried out. Results: Only one mAb and the polyclonal antibody showed a strong reaction band against rMSP10 and no cross reaction bands against non-infected RBC. Results by WB showed the presence of an approximately 70 kDa band in purified schizonts and rings stages parasites from 1 to 12 hours post-invasion, the binding of this antibody to mature schizonts was corroborated by IFA. Conclusion: In conclusion, we have identify one mAb and a polyclonal antisera capable to detect pfMSP10 protein in P. falciparum parasites. GIA in vitro assays are underway using Peruvian P. falciparum isolates in order to evaluate whether these antibodies are capable of binding to erythrocytes and inhibit the invasion of merozoites into erythrocytes. http://dx.doi.org/10.1016/j.ijid.2016.11.365 20.175 Autogenous vaccine field trial for epizootic hemorrhagic disease virus and bluetongue virus does not result in high titer to homologous virus serotypes S.M. Wisely a,∗ , K. Sayler b a
University of Florida, Department of Wildlife Ecology, Gainesville, FL - FLORIDA/US b University of Florida, Wildlife Ecology & Conservation, Gainesville/US Purpose: Epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) are vector-borne viruses that cause significant morbidity and mortality in ruminants worldwide. In North America, massive mortality events in wild and captive white-tailed deer (Odocoileus virgineanus) and elk (Cervus elaphus) occur in multi-year cycles. The captive cervid industry commonly uses commercially-produced, autogenous killed vaccines to protect animals from this complex of viruses, yet studies of efficacy and safety are lacking. The purpose of this study was to test the ability of a popular, commercially available autogenous vaccine to initiate and sustain antibody production in a herd of captive white tailed deer. Methods & Materials: We administered an autogenous killed virus vaccine according to manufacturer’s protocol in a population of captive white-tailed deer. The vaccine contained isolates of EHDV-1, EHDV-2, EHDV-6 and BTV-17 prepared from moribund white-tailed deer from Texas. Blood was drawn 45 days prior to the first vaccination, at the time of vaccine administration and at booster 30 days later for 25 vaccinated and 25 control white-tailed deer, which were housed outdoors in 1 acre pens. Serological assays were conducted to measure antibody titers to EHDV serotypes 1, 2 and 6 and BTV. Results: One month after vaccination, we found that young animals in the study did not produce elevated antibody titers relative to the baseline for any of the EHDV serotypes. All values were the same or lower than the baseline values measured prior to vaccination. No side effects from the vaccination were observed in any of the deer.
150
Abstracts / International Journal of Infectious Diseases 53S (2016) 4–163
Conclusion: The commercially prepared vaccine did not induce a robust, measurable humoral response in captive deer. Killed virus vaccines have previously been shown to provide effective protection from BTV in sheep. Efficacy of autogenous vaccines depends on multiple factors including the similarity of viruses in the vaccine to the wild-type strain, the use of a proper adjuvant to induce immune response, and purity of the vaccine. Vaccine makers must conduct proper testing of purity and efficacy of vaccine prior to offering its project to the market, and cervid owners are advised to test for antibody production if they are going to use autogenous vaccines. http://dx.doi.org/10.1016/j.ijid.2016.11.366 20.181 The changing patterns of Dengue and malarial infections -study from a Hospital in Mumbai India A. Barua ∗ , M.E. Yeolekar K J Somaiya Medical College and Hospital, Internal Medicine, Mumbai/IN Purpose: A definite change in the trend of Dengue and malarial infections, their clinical features and outcomes has been noticed recently. The present study, in a Mumbai hospital, during three consecutive monsoons, was carried out,to observe and compare the changing patterns of Dengue and malarial infections, in Mumbai, India. Methods & Materials: A comparative, retrospective cross sectional study of Dengue, Malaria and their coinfections was carried out, during three consecutive monsoons (June to November;20132015) in a Mumbai hospital. Febrile patients, during this period, were investigated for both Dengue and Malaria simultaneously. Elisa (NS-1/IgM) and peripheral smear examination was done to confirm Dengue and Malaria, respectively. Clinical comparison of signs and symptoms, severity and outcomes was systematically carried out. Results: During 2013,of the diagnosed acute febrile cases,41 were Malaria,39 being P.vivax and 2 mixed Malaria.52 cases of Dengue were confirmed.2014 saw a total of 55 malaria cases,23 being P.falciparum and 16 cases of P.vivax and mixed Malaria each. During the year,84 Dengue cases were detected. 2015 saw a surge of acute febrile illnesses.117 cases were of Malaria, 107 being P.vivax and only ten positive for P.falciparum. No mixed Malaria cases were encountered; whereas Dengue cases escalated to 206. During 2014, 16 (10.25%) coinfection (Dengue and Malaria) cases were noted, whereas in 2015, 28(6.7%)were coinfection cases. No coinfection cases were observed during 2013. Mortality during 2013 and 2014 were three each, all being P.vivax during 2013 whereas one was coinfection and two malarial deaths in 2014. Recovery was total in 2015. Conclusion: Within the three consecutive years, it was observed that Dengue cases exceeded Malaria, as a major cause of monsoon related febrile illnesses. Within the malarial infections, P.falciparum appears to be on the decline. P.vivax has increased in incidence and severity, thus not considered benign any longer. Focussed malaria control probably led to fewer malaria cases. Dengue formed the largest group, the surge being probably related to increased Aedes breeding sites. Changing clinical trends require close monitoring. Enhanced surveillance and public health measures can contribute to better disease control. http://dx.doi.org/10.1016/j.ijid.2016.11.367
20.182 West Nile virus neuroinvasive disease: The first confirmed case in Bulgaria M. Baymakova a,∗ , I. Trifonova b , E. Panayotova b , S. Dakova a , M. Pacenti c , L. Barzon c , E. Lavezzo c , K. Ramshev a , K. Plochev a , I. Christova a a
Military Medical Academy, Sofia/BG National Center of Infectious and Parasitic Diseases, Sofia/BG c University of Padova, Padua/IT b
Purpose: The first confirmed human case of WNV infection in Bulgaria was presented as a West Nile neuroinvasive illness with fatal outcome in a Bulgarian elderly man. Methods & Materials: For the etiological diagnosis of WNV infection specific serological tests were applied for detection of IgM in CSF and IgM and IgG in serum. WNV RNA was detected by real-time RT-PCR. Full genome sequencing was performed. Results: In the summer 2015, a 69-year old man with cardiovascular disorder and a history of mosquito bites and no recent travels outside Bulgaria, developed a febrile syndrome, tremor, and weakness, followed by neurological disturbances with coma and lethal outcome. CSF examination showed mild lymphocytic pleocytosis. WNV-specific IgM antibodies were detected in CSF and WNV-specific IgM and IgG antibodies were found in serum, WNV RNA was detected in a urine sample. Sequencing of the full viral genome and phylogenetic analysis demonstrated that the virus belonged to Southern-European WNV lineage 2 clade and had high sequence similarity with WNV strains circulating in Greece and in Hungary. Conclusion: This case report demonstrates the presence of WNV lineage 2 in Bulgaria and supports public health interventions for vector control and prevention of WNV transmission because of the risk of severe neuroinvasive disease and the high mortality rate, especially in elderly patients with co-morbidity. http://dx.doi.org/10.1016/j.ijid.2016.11.368 20.183 Severe clinical forms of Mediterranean Spotted Fever: A case series from an endemic area in Bulgaria M. Baymakova a,∗ , L. Pekova b , K. Plochev a , P. Parousheva b a b
Military Medical Academy, Sofia/BG Stara Zagora University Hospital, Stara Zagora/BG
Purpose: The aim of this study was to describe clinical, epidemiological and laboratory characteristics in patients with severe forms of Mediterranean spotted fever admitted to Bulgarian university hospital in endemic region. Methods & Materials: A retrospective study was conducted at the Department of Infectious Diseases, Stara Zagora University Hospital (Southeastern Bulgaria) between April 2015 and June 2016. For the analyzed period 54 cases had clinical and laboratory data for Mediterranean spotted fever (MSF). Raoult diagnostic criteria were used for the evaluation of severity. For the etiological diagnosis serological tests were applied. MSF-specific IgM and IgG antibodies were detected in serum by indirect immunoenzyme assay (ELISA IgG/IgM, Vircell, Spain). Rickettsia conorii ELISA IgG Sensitivity 85%, Specificity 100% and Rickettsia conorii ELISA IgM Sensitivity 94%, Specificity 95%. Statistical analyze was done by MS Excel 2007 and SPSS Statistics, version 19.0.
20.173 Epitiope based DNA vaccine design using epitopes predicted from Zika virus polyprotein P.S. Slathia ∗ , P. Sharma Shri Mata Vaishno Devi University, Biotechnology, Jammu, J&K/IN Purpose: Recent outbreaks of Zika virus have made the need for developing a vaccine imperative. The polyprotein sequences of the virus have been used for finding epitopes and designing an epitope based DNA vaccine using bioinformatics tools has been attempted in this study. Methods & Materials: NCBI Protein database was used as a source of polyprotein sequences of Zika virus. The sequences were studied for conservancy by MegAlign tool of DNASTAR Lasergene 8.0. NetMHCPan, NetMHCIIPan and IEDB analysis resource tools were used for finding Class I MHC binding peptides, Class II MHC binding peptides and B cell epitopes respectively. From the pool of binding peptides for MHC I CTL epitopes were predicted by CTLPred and NetCTL servers. The selected epitopic peptides were analyzed for similarity to human protein by BLASTp tool. AlgPred server was used for finding out the presence of allergenic peptides. The peptides were joined in tandem differently for MHC I, MHC II and B cell epitopes to generate three peptide sequences. The peptide properties were predicted by ProtParam of ExPasy tools. These peptides were reverse translated using Human Codon Usage table by Sequence Manipulation Suite. These gene sequences were then separately cloned into pVAC vector and the vector map was generated PlasMapper server. Results: 114 full length sequences of Zika virus polyprotein were retrieved; partial sequences were omitted for the study. The alignments revealed high degree of conservancy among the sequences and therefore consensus sequence was used for predictions. The MHC I & II peptides that were selected had no similarity to human protein sequences, were represented only once in the final sequence, were the strongest binders for the respective allele and had no allergenic properties. For the B cell epitopes only sequential or linear epitopes were predicted and the epitopes in the final sequence had all the properties as mentioned for MHC I & II except for allelic specificity. Cloning using human codons ensures the expression in humans. Conclusion: The DNA vaccine vector generated in the study has been rationally designed using existing principles of epitope based vaccine design and could be used after lab validation studies. http://dx.doi.org/10.1016/j.ijid.2016.11.364 20.174 Evaluation of monoclonal antibodies against pfMSP10 for the prospective use on in vitro growth inhibition assays of peruvian P. falciparum isolates E. Villasis a,∗ , J. Bendezu a , K. Garro a , O. Nolazco a , K. Torres b , B. Infante a , D. Gamboa b , J. Vinetz c a
UNIVERSIDAD PERUANA CAYETANO HEREDIA, CIENCIAS CELULARES Y MOLECULARES, Lima/PE b UNIVERSIDAD PERUANA CAYETANO HEREDIA, LIMA, Lima/PE c University of California, San Diego, San Diego, CA/US Purpose: The aim of this study is to evaluate monoclonal antibodies (mAb) generated against pfMSP10 (Merozoite surface
149
protein 10) and their functional role to inhibit the invasion of Peruvian P. falciparum isolates into red blood cells (RBC) by using the Growth inhibition Assay (GIA) in vitro Methods & Materials: Seven mAb and one polyclonal antibody were synthetized and evaluated by Western Blot (WB) against the recombinant MSP10 protein (rMSP10). Synchronized and purified schizonts from P. falciparum 3D7 and their concentrated supernatant were obtained by ultrafiltration. Detection of pfMSP10 protein was also evaluated in synchronized ring stage of P. falciparum cultures from 1 to 12 hours post-invasion. IFA assays were also carried out. Results: Only one mAb and the polyclonal antibody showed a strong reaction band against rMSP10 and no cross reaction bands against non-infected RBC. Results by WB showed the presence of an approximately 70 kDa band in purified schizonts and rings stages parasites from 1 to 12 hours post-invasion, the binding of this antibody to mature schizonts was corroborated by IFA. Conclusion: In conclusion, we have identify one mAb and a polyclonal antisera capable to detect pfMSP10 protein in P. falciparum parasites. GIA in vitro assays are underway using Peruvian P. falciparum isolates in order to evaluate whether these antibodies are capable of binding to erythrocytes and inhibit the invasion of merozoites into erythrocytes. http://dx.doi.org/10.1016/j.ijid.2016.11.365 20.175 Autogenous vaccine field trial for epizootic hemorrhagic disease virus and bluetongue virus does not result in high titer to homologous virus serotypes S.M. Wisely a,∗ , K. Sayler b a
University of Florida, Department of Wildlife Ecology, Gainesville, FL - FLORIDA/US b University of Florida, Wildlife Ecology & Conservation, Gainesville/US Purpose: Epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) are vector-borne viruses that cause significant morbidity and mortality in ruminants worldwide. In North America, massive mortality events in wild and captive white-tailed deer (Odocoileus virgineanus) and elk (Cervus elaphus) occur in multi-year cycles. The captive cervid industry commonly uses commercially-produced, autogenous killed vaccines to protect animals from this complex of viruses, yet studies of efficacy and safety are lacking. The purpose of this study was to test the ability of a popular, commercially available autogenous vaccine to initiate and sustain antibody production in a herd of captive white tailed deer. Methods & Materials: We administered an autogenous killed virus vaccine according to manufacturer’s protocol in a population of captive white-tailed deer. The vaccine contained isolates of EHDV-1, EHDV-2, EHDV-6 and BTV-17 prepared from moribund white-tailed deer from Texas. Blood was drawn 45 days prior to the first vaccination, at the time of vaccine administration and at booster 30 days later for 25 vaccinated and 25 control white-tailed deer, which were housed outdoors in 1 acre pens. Serological assays were conducted to measure antibody titers to EHDV serotypes 1, 2 and 6 and BTV. Results: One month after vaccination, we found that young animals in the study did not produce elevated antibody titers relative to the baseline for any of the EHDV serotypes. All values were the same or lower than the baseline values measured prior to vaccination. No side effects from the vaccination were observed in any of the deer.
150
Abstracts / International Journal of Infectious Diseases 53S (2016) 4–163
Conclusion: The commercially prepared vaccine did not induce a robust, measurable humoral response in captive deer. Killed virus vaccines have previously been shown to provide effective protection from BTV in sheep. Efficacy of autogenous vaccines depends on multiple factors including the similarity of viruses in the vaccine to the wild-type strain, the use of a proper adjuvant to induce immune response, and purity of the vaccine. Vaccine makers must conduct proper testing of purity and efficacy of vaccine prior to offering its project to the market, and cervid owners are advised to test for antibody production if they are going to use autogenous vaccines. http://dx.doi.org/10.1016/j.ijid.2016.11.366 20.181 The changing patterns of Dengue and malarial infections -study from a Hospital in Mumbai India A. Barua ∗ , M.E. Yeolekar K J Somaiya Medical College and Hospital, Internal Medicine, Mumbai/IN Purpose: A definite change in the trend of Dengue and malarial infections, their clinical features and outcomes has been noticed recently. The present study, in a Mumbai hospital, during three consecutive monsoons, was carried out,to observe and compare the changing patterns of Dengue and malarial infections, in Mumbai, India. Methods & Materials: A comparative, retrospective cross sectional study of Dengue, Malaria and their coinfections was carried out, during three consecutive monsoons (June to November;20132015) in a Mumbai hospital. Febrile patients, during this period, were investigated for both Dengue and Malaria simultaneously. Elisa (NS-1/IgM) and peripheral smear examination was done to confirm Dengue and Malaria, respectively. Clinical comparison of signs and symptoms, severity and outcomes was systematically carried out. Results: During 2013,of the diagnosed acute febrile cases,41 were Malaria,39 being P.vivax and 2 mixed Malaria.52 cases of Dengue were confirmed.2014 saw a total of 55 malaria cases,23 being P.falciparum and 16 cases of P.vivax and mixed Malaria each. During the year,84 Dengue cases were detected. 2015 saw a surge of acute febrile illnesses.117 cases were of Malaria, 107 being P.vivax and only ten positive for P.falciparum. No mixed Malaria cases were encountered; whereas Dengue cases escalated to 206. During 2014, 16 (10.25%) coinfection (Dengue and Malaria) cases were noted, whereas in 2015, 28(6.7%)were coinfection cases. No coinfection cases were observed during 2013. Mortality during 2013 and 2014 were three each, all being P.vivax during 2013 whereas one was coinfection and two malarial deaths in 2014. Recovery was total in 2015. Conclusion: Within the three consecutive years, it was observed that Dengue cases exceeded Malaria, as a major cause of monsoon related febrile illnesses. Within the malarial infections, P.falciparum appears to be on the decline. P.vivax has increased in incidence and severity, thus not considered benign any longer. Focussed malaria control probably led to fewer malaria cases. Dengue formed the largest group, the surge being probably related to increased Aedes breeding sites. Changing clinical trends require close monitoring. Enhanced surveillance and public health measures can contribute to better disease control. http://dx.doi.org/10.1016/j.ijid.2016.11.367
20.182 West Nile virus neuroinvasive disease: The first confirmed case in Bulgaria M. Baymakova a,∗ , I. Trifonova b , E. Panayotova b , S. Dakova a , M. Pacenti c , L. Barzon c , E. Lavezzo c , K. Ramshev a , K. Plochev a , I. Christova a a
Military Medical Academy, Sofia/BG National Center of Infectious and Parasitic Diseases, Sofia/BG c University of Padova, Padua/IT b
Purpose: The first confirmed human case of WNV infection in Bulgaria was presented as a West Nile neuroinvasive illness with fatal outcome in a Bulgarian elderly man. Methods & Materials: For the etiological diagnosis of WNV infection specific serological tests were applied for detection of IgM in CSF and IgM and IgG in serum. WNV RNA was detected by real-time RT-PCR. Full genome sequencing was performed. Results: In the summer 2015, a 69-year old man with cardiovascular disorder and a history of mosquito bites and no recent travels outside Bulgaria, developed a febrile syndrome, tremor, and weakness, followed by neurological disturbances with coma and lethal outcome. CSF examination showed mild lymphocytic pleocytosis. WNV-specific IgM antibodies were detected in CSF and WNV-specific IgM and IgG antibodies were found in serum, WNV RNA was detected in a urine sample. Sequencing of the full viral genome and phylogenetic analysis demonstrated that the virus belonged to Southern-European WNV lineage 2 clade and had high sequence similarity with WNV strains circulating in Greece and in Hungary. Conclusion: This case report demonstrates the presence of WNV lineage 2 in Bulgaria and supports public health interventions for vector control and prevention of WNV transmission because of the risk of severe neuroinvasive disease and the high mortality rate, especially in elderly patients with co-morbidity. http://dx.doi.org/10.1016/j.ijid.2016.11.368 20.183 Severe clinical forms of Mediterranean Spotted Fever: A case series from an endemic area in Bulgaria M. Baymakova a,∗ , L. Pekova b , K. Plochev a , P. Parousheva b a b
Military Medical Academy, Sofia/BG Stara Zagora University Hospital, Stara Zagora/BG
Purpose: The aim of this study was to describe clinical, epidemiological and laboratory characteristics in patients with severe forms of Mediterranean spotted fever admitted to Bulgarian university hospital in endemic region. Methods & Materials: A retrospective study was conducted at the Department of Infectious Diseases, Stara Zagora University Hospital (Southeastern Bulgaria) between April 2015 and June 2016. For the analyzed period 54 cases had clinical and laboratory data for Mediterranean spotted fever (MSF). Raoult diagnostic criteria were used for the evaluation of severity. For the etiological diagnosis serological tests were applied. MSF-specific IgM and IgG antibodies were detected in serum by indirect immunoenzyme assay (ELISA IgG/IgM, Vircell, Spain). Rickettsia conorii ELISA IgG Sensitivity 85%, Specificity 100% and Rickettsia conorii ELISA IgM Sensitivity 94%, Specificity 95%. Statistical analyze was done by MS Excel 2007 and SPSS Statistics, version 19.0.