| PREFACEMalaria is a disease caused by infection with protozoan parasites of the genus Plasmodium and transmitted by Anopheles mosquitoes. It is estimated that 2. 2 billion people were exposed to the threat of P. falciparum malaria, resulting in a conservative estimate of 515(rang 300 -660) million clinical attacks attributable to this parasite during 2002. At a regional level, most clinical events attributable to P. falciparum were concentrated in the African region (70% ) , but the highly populated South East Asia region contributed 25% of the world's clinical attacks in 2002. Vaccines targeting antigens expressed on the surface of the sexual stages of malaria parasites are considered a promising strategy for malaria control. These transmission blocking vaccines (TBVs) may effectively interrupt parasites, and may also prevent the spread of parasites that are resistant to other vaccines or drugs, thereby prolonging their effective life.Pvs25 and Pvs28, the plasmodium vivax zygote and ookinete surface proteins , which play a role in ookinete survival in the mosquito midgut, are considered to be important transmission blocking vaccine candidates. Although P. falciparum ookinete surface proteins are highly conserved, polymorphism has been detected in Pvs25 and Pvs28 molecules isolated from malaria — endemic areas. In the current study, it is only 5 amino acid substitutions in Pvs25. In contrast, a total of 22 amino acid substitutions were found in Pvs28. The antigenic diversi-ty is one of the most serious obstacles to developing effective malaria vaccine. The analysis of genetic polymorphism is the chiefly task before malaria vaccine is widely used in malaria epidemic regions.The previous reports showed that Pvs25 was highly conserved among Zhe-jiang and Hubei isolates, and Pvs28 had limit mutations. In this study, we assessed the levels of genetic diversity of Pvs25 and Pvs28 from Yunnan and Zhe-jiang isolates and evaluate the possible applicability of TBVs before the wide range applicability of Sal -1 based vaccines in China.MATERIALS AND METHODSStudy sites; Yunnan province is a malaria year - round transmission of P. vivax and P. falciparam. Zhejiang province is a seasonal transmission of P. vivax. The 31 and 24 of P. vivax isolates from Yunnan province and Zhejiang province were collected, respectively. P. vivax genomic DNA was extracted from dried filter paper blood spots which obtained by using QIAamp DNA mini kit. The full - length open reading frame of the Pvs25 and Pvs28 from P vivax genomic DNA was amplified with a polymerase chain reaction. The PCR products were directly sequenced. Computer - based algorithms were used to analyze neu-cleotide and amino acid sequences and the sequence alignments were done with Mac Vector software. Intxapopulation and interpopulation diversity indexes were analyzed by DnaSP version 4. 0 software.RESULTSl.The 31 and 41 sequences of Pvs25 and Pvs28 complete gene were obtained, respectively.2. Compared with Sal -1 Pvs25 sequence, there were 4 non - synonymous mutation sites ( C/A103, G/C289, T/C389, C/A391) and 5 kinds of haplotype fromYunnan isolates, (1)C - G - C - C ; (2)L - G -L - A ; (3)C -3. Compared with Sal -1 Pvs28 sequence, there were 7 non - synonymousmutations sites and 7 kinds of haplotypes from Yunnnan isolates, (1)G40 - C154 -T292 _ G313 _ (.346 _ Q419 _ C666 . (2)G40 _ A154 _ A292 _ (.313 _ G346 _ C419 C666.(3)T40 - A154 - T292 - A313 - G346 - C419 - C666;(4)G40 - A154 - T292 - G313 - G346 -C419 - G666 (5)G40 - A154 - T292 - G313 - C346 - C419 - G666 ;(6)G40 - C154 - T292 -G313 - T346 - C419 - C666; (DG40 - A154 - T292 - A313 - G346 - C419 - C666 0 There were 2 synonymous mutations sites, 2 non - synonymous mutations sites and 2 kinds of haplotypes from Zhejiang isolates, (J)G - C154 - i - G ' -G1 -4. The data of nucleotide diversity showed that the Pvs28 isolates from Yunnan was more polymorphic than Zhejiang( it =0.0044,0.0002). The nucleotide diversity of Pvs25 was 0.0014 of Yunnan isolates.5. Compared with reported previously, in Chinese Yunnan, Zhejiang andHubei malaria epedemic regions, L - E - T - Q and V - L - L - L -V106 — L116 - S140 - I223 were the predominant amino acid types of Pvs25 and Pvs28, respectively.DISCUSSIONIn this study, we detected that there were 4 non - synonymous mutation sites, 5 kinds of haplotypes in Pvs25 and 7 non - synonymous mutations sites, 7 kinds of haplotypes in Pvs28. It was indicated that Pvs25 was more conserved than Pvs28. Compared with reported previously, in Chinese Yunnan, Zhejiang and Hubei malaria epedemic regions, L - E - T - Q131 and V14 - L - L-V -L -b -1 were the predominant ammo acid types, respectively. The data of nucleotide diversity showed that the isolates from Yunnan was more polymorphic than Zhejiang.Dr. Sattabongkot Jetsumon has analyzed the relationship between the transmission - blocking activity of immune mouse serum and the genetic polymorphism of the Pvs25 and Pvs28. It clearly demonstrated that antibodies raised a-gainst Sal -1 based vaccines overcome the genetic diversity would not limit the efficacy of Pvs25 and Pvs28 based TBVs. Compared with the sequences of Pvs25 and Pvs28 from Thailand isolates ( GenBank?: AB091729 - AB091745), almostmutations sites of this study were included in the mutations of Thailand' s study, exception of one mutation of Pvs25 and two mutations of Pvs28. But those three mutations caused conserved amino acid substitutions. Though there were limited Pvs25 and Pvs28 mutations found in Yunnan, Zhejiang and Hubei isolates, it suggested that Sal -1 based vaccines can overcome the genetic polymorphism of Pvs25 and Pvs28 present in Chinese natural isolates of P. vivax.The mechanism resulted in Pvs25 and Pvs28 polymorphism may be two reasons. The mosquito entomological inoculation rate (EIR) is likely to be a major influence on the complexity of parasites found in individuals and consequently in the community. A high EIR in the year -round malaria transmission region, allowing continual inoculations of parasites, would be more chances for sexual antigen mutation than in the seasonal malaria transmission region. The mosquito vector maybe the another reason. The mosquito midgut environments and the species of mosquito maybe contribute to the complexity of parasites.The recombinant proteins expressed in yeast elicited effective transmission - blocking antibodies in mice. The DNA vaccine based on Pvs25 and Pvs28 e-licited high titers of specific antibodies that effectively blocked transmission of the parasite. In this study, we assess the levels of genetic diversity of Pvs25 and Pvs28 within and between populations from two regions and evaluate the possible applicability of TBVs.CONCLUSION1. It is reinforced that the transmission blocking vaccine candidate Pvs25 is highly conserved and is less polymorphic than Pvs28.2. The genetic polymorphism of Pvs25 and Pvs28 has geographic difference between year - round transmission regions and seasonal transmission regions. The isolates from Yunnan province are more polymorphic than those from Zhejiang province.3. Though geographic genetic diversity is obviously distinct, the predominant amino acid type of Pvs25 is only one type, as well as the main amino acid type of Pvs28. |
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