High yield expression of leptospirosis vaccine candidates LigA and LipL32 in the methylotrophic yeast Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>Leptospirosis, a zoonosis caused by <it>Leptospira </it>spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in <it>Escherichia coli </it>have demonstrated promising results, albeit with variable efficacy. <it>Pichia pastoris </it>is an alternative host with several advantages for the production of recombinant proteins.</p> <p>Results</p> <p>The vaccine candidates LigANI and LipL32 were cloned and expressed in <it>P. pastoris </it>as secreted proteins. Large-scale expression resulted in a yield of 276 mg/L for LigANI and 285 mg/L for LipL32. The recombinant proteins were glycosylated and were recognized by antibodies present in the sera of patients with severe leptospirosis.</p> <p>Conclusions</p> <p>The expression of LigANI and LipL32 in <it>P. pastoris </it>resulted in a significant increase in yield compared to expression in <it>E. coli</it>. In addition, the proteins were secreted, allowing for easy purification, and retained the antigenic characteristics of the native proteins, demonstrating their potential application as subunit vaccine candidates.</p

Hepatitis A virus prevalence: Portuguese first results of low endemicity

The prevalence of the hepatitis A virus in a population determines the degree of morbidity associated with this illness, that is, the higher the morbidity, the lower the prevalence. This study aims to obtain the prevalence of total antibody to the hepatitis A virus in children, 5 and 8 years of age, and in adolescents, 14 years of age. The study was based on two samples: the serum of 64 healthy five-year-olds and 76 healthy eight-year-olds living in the proximity of the São Marcos Hospital in Braga and the serum of 311 adolescents, aged 14, from a total population of 536 adolescents attending schools in Braga, North of Portugal. The samples were collected in 1999 for the adolescent group, in 2000 and 2001 for the group of the five-year-olds and in 2002 and 2003 for the group of the eight-year-olds. None of the persons involved had been vaccinated with the hepatitis A virus vaccine. The Enzyme Linked Fluorescent Assay method was used to measure the serum total antibody to the hepatitis A virus. The prevalence of total antibody to the hepatitis A virus was 1.6% at 5 years of age, [95% confidence intervals (CI), 0-4.7%]; 3.9% at 8 years of age, [95% CI, 0-8,4%]; and 32.5% at 14 years of age +/- 6 months, [95% CI, 27.3-37.7%]. The prevalence of total antibody to the hepatitis A virus in this population revealed lower natural immunity. The results obtained for the five and eight-year-olds were the first Portuguese results of low endemicity to the hepatitis A virus

The use of halloysite clay and carboxyl-functionalised multi-walled carbon nanotubes for recombinant LipL32 antigen delivery enhanced the IgG response

We studied the feasibility of using halloysite clay nanotubes (HNTs) and carboxyl-functionalised multi-walled carbon nanotubes (COOH-MWCNTs) as antigen carriers to improve immune responses against a recombinant LipL32 protein (rLipL32). Immunisation using the HNTs or COOH-MWCNTs significantly increased the rLipL32-specific IgG antibody titres (p < 0.05) of Golden Syrian hamsters. None of the vaccines tested conferred protection against a challenge using a virulent Leptospira interrogans strain. These results demonstrated that nanotubes can be used as antigen carriers for delivery in hosts and the induction of a humoral immune response against purified leptospiral antigens used in subunit vaccine preparations

Evidência do cão como reservatório da leptospirose humana: isolamento de um sorovar, caracterização molecular e utilização em inquérito sorológico Evidence of dog as a reservoir for human leptospirosis: a serovar isolation, molecular characterization and its use in a serological survey

A leptospirose canina é conhecida como enfermidade de Stuttgard desde 1898, sendo os cães, depois dos roedores, considerados como a segunda principal fonte de infecção para o homem. O isolamento de um sorovar patogênico da urina de um cão, laboratorial e clinicamente identificado como tendo leptospirose, e sua utilização para testar amostras de soro de casos de leptospirose humana e canina, evidenciou a sua importância no ecossistema da região sul do Brasil. Os resultados do teste de soroaglutinação microscópica indicaram que 100% das amostras de soro humano de 12 pacientes do banco de soro de 2001 do Centro de Controle de Zoonoses, que haviam reagido com títulos que variaram de 25 a 3.200 para o sorovar canicola, e 72% das amostras de 105 soros caninos do mesmo banco de soro, também reagiram contra o novo isolado. O título médio e mediana dos soros humanos testados com a bateria de antígenos recomendada pela OMS, foi respectivamente 630 e 100, ao passo que os testados com o isolado foi de 1.823 e 400. Nos soros caninos, os títulos foram respectivamente de 347 e 100 para a bateria e de 1.088 e 200 para o isolado.<br>Canine leptospirosis has been known as Stuttgart disease since 1898, and dogs are considered to be the second principal source of infection in man. The isolation of a pathogenic serovar from dog urine that was diagnosed clinically and laboratorial as having leptospirosis and its utilization to test serological samples of human and canine cases of leptospirosis, has demonstrated its importance to the ecosystem of the southern region of Brazil. The results of the serological microscopic agglutination test indicated that 100% of human serum samples from 12 patients from the serum bank of 2001 at the Center for Control of Zoonoses, that had titers between 25 and 3,200 with the canicola serovar, and 72% of 105 canine serum samples from the same serum bank, also reacted with the new isolate. The mean and median titers of the human serum samples tested with the battery of antigens recommended by WHO was 630 and 100 respectively, and when tested with the isolate it was 1,823 and 400. In the dog sera, the values were respectively 347 and 100 with the battery, and 1,088 and 200 with the isolate