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Cytokine 52 (2010) 151–155

Contents lists available at ScienceDirect

Cytokine journal homepage: www.elsevier.com/locate/issn/10434666

Short Communication

DNA sequencing of 13 cytokine gene fragments of Aotus infulatus and Saimiri sciureus, two non-human primate models for malaria F.A. Alves a,f,⇑, M.T. Souza a, E.C. Gonçalves b, M.P.C. Schneider b, A.M. Marinho e, J.A.P.C. Muniz d, S.P. Fragoso c, M.A. Krieger c, S. Goldenberg c, C.T. Daniel-Ribeiro a, L.J.M. Carvalho a,1 a Laboratory of Malaria Research, Laboratory for Malaria Research, Instituto Oswaldo Cruz and Center for Malaria Research and Training (CPD-Mal), Fiocruz and SVS, Rio de Janeiro, Brazil b Laboratory of DNA Polymorphisms, Universidade Federal do Pará, Belém, Brazil c Molecular Biology Institute of Paraná, Fiocruz, Curitiba, Brazil d National Primate Center, Belém, Brazil e Cecal, Fiocruz, Rio de Janeiro, Brazil f Laboratory of Immunology, Universidade Federal do Pará, Belém, Brazil

a r t i c l e

i n f o

Article history: Received 25 March 2010 Received in revised form 21 July 2010 Accepted 15 September 2010

Keywords: Aotus Saimiri Immune system Cytokines Malaria

a b s t r a c t Aotus and Saimiri are non-human primate models recommended by the World Health Organization for experimental studies in malaria, especially for vaccine pre-clinical trials. However, research using these primates is hindered by the lack of specific reagents to evaluate immune responses to infection or vaccination. As a step toward developing molecular tools for cytokine expression studies in these species, primer pairs for 18 cytokine gene fragments were designed based on human DNA sequences and used to amplify the corresponding genes in Aotus infulatus and Saimiri sciureus genomic DNA samples. IFNc, TNFa, LTA, IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, CSF2 and TGFb2 gene fragments were amplified and sequenced. Primer pairs for IL8, IL17, IL18, IL27 and MIF failed to generate amplification products. When compared to the available corresponding human and non-human primate sequences, most – except IL3 and IL4 – showed identity degrees above 90%. Small variations in sequence can help to explain the failure to amplify certain genes or the amplification only at lower annealing temperatures as compared to human DNA samples for several primer pairs. The sequences made available provide the basis for designing molecular tools such as primers for real time PCR specific for A. infulatus and/or S. sciureus. The nucleotide sequences reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers DQ985386 to DQ985389, DQ989356 to DQ989369, FJ89020 to FJ89024 and FJ89029. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Non-human primates of the genera Aotus and Saimiri are experimental models recommended by the World Health Organization for research in malaria [1–4]. The main advantage of these models is their unique ability to develop reproducible parasitemias when inoculated with blood stages or even sporozoites of the human plasmodial species Plasmodium falciparum and P. vivax, being the closest animal models to the human infection, making them particularly useful in pre-clinical trials of potential malaria vaccines, as ⇑ Corresponding author. Address: Laboratory of Immunology, Instituto de Ciências Biológicas, Universidade Federal do Pará – UFPA, Guamá, Belém, PA 66075-110, Brazil. Tel./fax: +55 21 3865 8145. E-mail address: falves@ufpa.br (F.A. Alves). 1 Current address: La Jolla Bioengineering Institute, La Jolla, CA. 1043-4666/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2010.09.004

well for studies on pathogenesis [5,6,24,25]. However, some factors restrict the use of these neotropical primates in malaria research, such as the limited availability of research-quality animal colonies and the lack of specific reagents and molecular tools allowing, for instance, reliable evaluation of immune responses. In some cases, immunological reagents for human molecules can be used although sensitivity is usually low [7,8]. We have previously used anti-human IgG antibodies to assess humoral immune responses in Saimiri and Aotus, but sensitivity was greatly improved by using an in-house rabbit anti-Saimiri IgG [9,10]. Molecular studies are subjected to similar restrictions [11] as small variations in nucleotide sequences can interfere with amplification or hybridization reactions, and research effort has been directed to sequencing individual genes of interest such as cytokines [12–14], immunoglobulins [15,16], Duffy blood group [17], MHC [18], TCR [19–21] and toll-like receptors [22], in some cases generating tools for quanti-


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tative evaluation of mRNA expression [12,23]. The development of reliable molecular primers or probes to detect expression of cytokines and other immune genes of Aotus and Saimiri will require sequencing the genes of interest for these species. In the present paper, we sought to describe and analyze partial sequences of 18 Aotus and Saimiri cytokine genes.

Table 1 Human oligonucleotide sequences used for amplification and sequencing of Aotus and Saimiri gene fragments. Gene

Primer sense

Primer sequence

Tm (°C)

IFNc

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

50 -CAGGTCATTCAGATGTAGC-30 50 -CACCGAATAATTAGTCAGC-30 50 -GACAGCAGAGGACCAGCTAAGAG-30 50 -GGCCGATCACTCCAAAGTGCAG-30 50 -ATGACACCACCTGAACGTCT-30 50 -CGAAGGCTCCAAAGAAGACA-30 50 -TGTACAGGATGCAACTCCTGTC-30 50 -CTTAAATGTGAGCATCCTGGTGAG-30 50 -CTTCGAAGGCCAACCCTG-30 50 -AAGGTGAAATGCTTCTGGTC-30 50 -AGCTTCTCCTGATAAACTAATTGCC-30 50 -CAGCAAAGATGTCTGTTACGG-30 50 -ACTGAAGAAATCTTTCAGGG-30 50 -TCGGTGTTCATTACACCA-30 50 -GAACCTTCCAAAGATGGC-30 50 -CAAATCTGTTCTGGAGGT-30 50 -CAGCAAATGAAGGATCAGCTGGACAACT-30 50 -ACAGCGCCGTAGCCTCAGCCTGAGGGTCTTC-30 50 -CCAGACAAACTCTAGAATTTTACCCTT-30 50 -CCTGCATCAGCTCATCAATAACTGCC-30 50 -TGCAATGGCAGCATGGTATGG-30 50 -ACCTTGTGCGGGCAGAATCC-30 50 -AGAGAAAGGCTAAAGTTCTCTG-30 50 -CCTGGAGGTCAAACATTTCTG-30 50 -CCAAAGATTTAACATCTCCAACCCA-30 50 -GCAAATCTTGCTTCTAGTTCTTCAC-30 50 -GAGTGCTAAAGAACTTAGATGTCAG-30 50 -AAACTTCTCCACAACCCTC-30 50 -CCGCAATGAGGACCCTGAGAG-30 50 -GAAAATGGTTACGATGTGAAA-30 50 -AAAACCTGGAATCAGATTACT-30 50 -CTCTACAGTCAGAATCAGTCA-30 50 -TTGGAGCTCGTCTTATCT-30 50 -TAGCTGGCGAGGACCAGA-30 50 -TTCCTCGCAGTACATCGC-30 50 -GTCTCTAAACCGTTTATTTCTCCC-30

58.1

TNFa

2. Material and methods LTA

2.1. Animals Saimiri sciureus and Aotus infulatus monkeys were bred and housed at the National Primate Center, SVS, in Belém, Pará (Saimiri and Aotus), and at the Department of Primatology, Fiocruz, Rio de Janeiro (Saimiri), Brazil. All animals used in this work were born in captivity. Protocols involving animal handling and blood withdrawal were approved by the Ethical Committee for Animal Use of Fiocruz.

IL2 IL3 IL4 IL5 IL6 IL10

2.2. Blood samples for DNA extraction

IL12

Blood samples were obtained by femoral venous puncture. Density gradient separation using 1077 Ficoll Hypaque (Sigma) was carried out to separate PBMCs. Aotus and Saimiri total genomic DNA was isolated and extracted following the phenol–chloroform protocol [26]. Human genomic DNA, used as a positive control for amplification, was obtained from PBMC from donors in the Laboratory of Malaria Research, Fiocruz.

IL13

2.3. Genes of interest and primer design

IL18

A panel of 18 cytokine genes was selected for sequencing: IFNc, TNFa, LTA (TNFb), IL2, IL3, IL4, IL5, IL6, IL8, IL10, IL12, IL13, IL17, IL18, IL27, CSF2, TGFb2 and MIF. We chose to use human sequences obtained from the GenBank™ (http://www.ncbi.nlm.nih.gov/entrez/) as templates to design PCR primers using the web-free PerlPrimer™ software (http://perlprimer.sourceforge.net/). The criteria used to design primers for amplification and sequencing were as follows: (i) primers 18 to 25 bp; (ii) melting temperature (Tm) ranging from 50 to 70 °C; (iii) Tm variation between forward and reverse primers not exceeding 2 °C; (iv) amplicons not exceeding 1000 base pairs (bp); and (v) amplicons preferentially including two exons. Forward and reverse primer sequences are shown in Table 1. Once each primer sequence (forward and reverse) was defined, primers were synthesized (Imprint Genetics Corp, Sao Paulo, Brazil). 2.4. Polymerase chain reaction, sequencing, alignment and sequence edition The best annealing temperature (Tm) for each primer pair was defined by running temperature gradient PCRs. Aotus and Saimiri as well as human genomic DNA were denatured at 94 °C for 5 min followed by 30 cycles with temperatures ranging from 50 °C to 65 °C for 1 min, 72 °C for 5 min, and final cycle at 72 °C for 1 min. The amplicons were run in 1% agarose gel, stained with ethidium bromide and visualized on an UV transilluminator. The annealing temperature chosen for the next steps for each gene was one at the higher temperature range providing a clear band for both primate species without associated unspecific bands, and are shown on Table 1. After defining the Tm for each primer pair, PCR reactions were carried out to amplify the respective gene fragments from three Aotus and three Saimiri individuals, as well as one human control DNA sample. PCR products were purified using GFX kit™ (GE Healthcare, UK) and sequenced in a ABI3730 analyzer

CFS2 TGFb IL8 IL17

IL27 MIF

59.3 54.0 58.1 58.1 59.3 58.1 58.1 62.7 62.1 59.3 59.3 54.0 54.0* 54.0* 54.0* 54.0* 54.0*

Tm: optimal annealing temperature. * Tm for human DNA (Aotus and Saimiri DNA not amplified).

(Applied Biosystems, Carlsbad, CA) by the chain terminator method using the same primers used for PCR [26,27]. The alignment and edition of sequences were carried out using the Bioedit™ software (www.mbio.ncsu.edu/BioEdit/). 3. Results 3.1. Primer design and amplification Out of the 18 primer pairs tested, 13 were able to amplify Saimiri and/or Aotus DNA fragments within the range of temperatures tested during gradient PCR: IFNc, TNFa, LTA, IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, CSF2 and TGFb. Fig. 1 shows the amplification products on gradient PCR agarose gels for some genes. The primer sets designed for IL8, IL17, IL18, IL27 and MIF were able to amplify human but not Aotus or Saimiri DNA. In addition, whereas all primer sets tested generated amplification products with human DNA in most or all the temperatures tested, in most cases Aotus and Saimiri DNA generated amplicons in the lower temperature range, suggesting limited primer complementarity and indicating the probable presence of small differences in nucleotide sequence in the target regions. In some cases amplicons were obtained for one but not the other monkey species: IL10 was amplified and sequenced with Saimiri but not with Aotus DNA, and vice versa for IL5 (Table 2). The lower specificity at lower temperatures was also revealed by the appearance of unspecific bands in some cases, as for IL13 (Fig. 1).


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Table 2 Identity degree (%) of nucleotide sequences of gene fragments from Aotus infulatus and Saimiri sciureus compared with human and other non-human primate orthologous sequences. Gene

IFNc

TNF

LTA

IL2

IL3

IL4

Fig. 1. Examples of temperature gradient PCRs performed with Aotus, Saimiri and human genomic DNA samples using the designed primer pairs for IL2, IL12 and IL13. The lowest and the highest temperatures tested are shown. Temperature intervals varied from 50 to 65 °C. The annealing temperatures selected for amplification of each gene fragment are shown (arrow).

3.2. Identity degree The selected Tm was used in PCR to generate amplicons for each of the 13 gene fragments with successful amplifications in the previous round. Clear amplification products were obtained with most of the 13 primer sets, IL5 being the exception and generating faint bands in the agarose gel (data not shown). Each amplification product was purified and sequenced, and nucleotide sequences were compared with human and non-human primate sequences deposited in GenBankÒ using BioEditÒ (Supplemental Fig. 1). The amplified DNA fragments of S. sciureus and A. infulatus showed overall elevated degrees of identity with the human sequences, only IL3 and IL4 showing less than 90% identity (Table 2). As expected, when comparing the obtained sequences with available sequences deposited for Saimiri or Aotus, identity was higher, usually in the 95–100% range. Nevertheless, in some cases such as IL4, differences between A. infulatus and other Aotus species reached close to 10%. Identity between S. sciureus and A. infulatus sequences was also high overall.

IL5

IL6

IL10

IL12B IL13

4. Discussion CSF2

In the present paper, we generated amplification products for 13 out of 18 cytokine genes from Saimiri and Aotus, using

Species (GenBankÒ access numbers)

A. infulatus

S. sciureus

A. infulatus  S. sciureus

Human (AF506749) S. sciureus (AF414102) A. lemurinus (AF097327) A. nancymaae (AF014512) A. nigriceps (AF097326) A. vociferans (AF014507) M. mulatta (AY376145) Human (AY066019) S. sciureus (AF294760) M. mulatta (NM_001047149) Human (AY070490) A. nancymaae (AY373461) M. mulatta (NM_001047148) Human (AF359939) S. sciureus (AF294755) A. lemurinus (U88364) A. nancymaae (U88361) A. nigriceps (U88363) A. vociferans (U88362) M. mulatta (U19847) Human (AF365976) M. mulatta (NM_001101734) Human (M23442) A. lemurinus (AF097321) A. nancymaae (AF014509) A. nigriceps (AF097320) A. vociferans (AF014504) M. mulatta (L26027) Human (AF353265) S. sciureus (AF294756) M. mulatta (XM_001047133) Human (AF372214) M. mulatta (NM_001042733) S. sciureus (AF294757) A. lemurinus (AF097323) A. nancymaae (AF014510) A. nigriceps (AF097322) A. vociferans (AF014505) Human (AF418271) S. sciureus (AF294758) A. lemurinus (AF097325) A. nancymaae (AF014511) A. nigriceps (AF097324) A. vociferans (AF014506) M. mulatta (DQ890063) Human (AF512686) M. mulatta (U19841) Human (AF377331) M. mulatta (NM_001032929) Human (AF373868) M. mulatta

93 98.4 99

92 99.2 98

99.2

97.2

96.4

99.2 99

98.4 98

92 94 95.3 91.2

91.2 90.1 99 89

95.2 100

96 99

96

97

98 99.5 100 100 100 100 97.9 87 91

97.4 99 99.5 99.5 99.5 99.5 97.4 81 89

99.5

87.4 90.1

92 93

91.1

91.4

92

91.4 91.4

92 92

85.2 91.4 98.2 92

87.4 – – –

96 94.1

95 93.1

98 95

99 94.1

95

94.1

99 98

98 97

– – –

89 98.1 93.3

91.1

– –

93.3 93.3

– 93 92 94 96.1

91.6 95 94 93 95

91.1 91

89 88.1

96.4

99

89.1

99

96.3 98

97 (continued on next page)


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Table 2 (continued) Gene

TGFb2

Species (GenBankÒ access numbers)

A. infulatus

S. sciureus

A. infulatus  S. sciureus

(NM_001032949) Human (AY438979) M. mulatta (LOC707540)

98 96

97.4 95.3

99

sequences of human genes as templates to design primers. The difficulty of amplification for genes that are in general well conserved among species, especially in primates, reinforces the need for the design and use of specific reagents to work with these models, since small differences in sequence can indeed cause difficulties or even preclude the proper annealing, resulting in failure to obtain amplified products. Our results also showed that even when a given primer pair was able to generate an amplification product, amplification was usually less efficient with Saimiri and/or Aotus than with human DNA, probably reflecting an imperfect match during annealing. Indeed, the effect of small sequence differences in the recognition of orthologous molecules in different species is well illustrated by studies showing that injection of recombinant human cytokines in non-human primates induce potent neutralizing antibody response against these cytokines, even when the homologies in both the peptide and nucleotide sequences are greater than 95% [7]. The differences in sequence and, potentially, in conformation can also explain the results obtained by Contamin and coworkers [28], who used a panel of 67 monoclonal antibodies of different manufacturers to define their reactivity against CD markers in Saimiri mononuclear cells. In some cases good reactivity was observed with a variation of intensity in staining depending on the clone used and the manufacturer, but in many cases no reactivity was detected. Likewise, Daubenberger and colleagues [29] tested 204 antibodies against various human CD markers in Aotus splenocytes and only 54 (28%) reacted. Out of these 54, only 22 (i.e. 11% of the total) were able to react also with rhesus and cynomolgus cells. These results altogether strengthen the rationale for obtaining specific reagents for monkeys. The lack of specific reagents and the need to better assess immune responses in the two main non-human primate models for malaria research, Saimiri and Aotus, has led to some effort by different groups to sequence, clone and characterize genes and/or proteins related to the immune system in these animals, particularly in different species of Aotus such as A. nancymaae, A. lemurinus griseimembra, A. nigriceps and A. vociferans. These works usually found high degrees of identity between these non-human primate and human sequences for the genes studied, such as cytokines (IL2, IL4, IL6, IL10, TNF and IFN) [13], CD45 [30], toll-like receptor 9 [22] and the Duffy-binding protein [17]. Lower degree of homology was observed when studying T cell receptors [19–21], immunoglobulin heavy and light chains [15,16] and MHC molecules [18]. Fewer works have been done with Saimiri genes, with emphasis on cytokines [12–14]. Some works went further using the sequences obtained to develop tools to detect and quantify the expression of the studied genes, such as competitive plasmids for semi-quantitative evaluation of mRNA expression of the Saimiri cytokines IL1, IL2, IL5, IL6, IL10, TNFa and IFNc [12] or primers for qRT-PCR detection of the Aotus cytokines IFNc, IL4, IL10 and LTA [23]. Expanding the number of gene sequences available for Saimiri and Aotus is important, especially with the increasing accessibility to methodologies such as microarrays and multiplex qRT-PCR, which allow fast and reliable analysis of the expression of multiple genes simultaneously.

We opted to directly sequence fragments of genomic DNA rather than to purify and clone mRNAs because, in addition to being simpler and faster, it decreases the possibilities of point mutations which may occur during cloning. The next step is to use the information gathered to design and validate primers that can be used for quantitative evaluation of cytokine mRNA expression in these primates. Because most primer pairs based on human sequences used in this work were able to amplify Saimiri and/or Aotus gene fragments, these primers themselves can be used a priori in expression studies using conventional RT-PCR. However, as discussed above, amplification with these primers was usually less efficient with S. sciureus and A. infulatus than with human DNA. Therefore, for quantitative experiments, it may be desirable to have primers with improved performance. The sequences of gene fragments comprising 13 molecules described here will be useful in the design of such primers and probes, circumventing potential restrictions imposed by the use of reagents derived from human or Old World primate sequences. In addition, the availability of both S. sciureus and A. infulatus sequences makes it possible to design primers and probes with sequences conserved in both genera, as well as to other species of Saimiri and Aotus whose sequences are available. This is particularly important given the observation of differences in sequences found between genera and even within different species of the same genus, for most genes studied. In this case, a primer set designed for one particular species may not work for another species in the same genus. More likely, primers designed for Saimiri may not work for Aotus, despite their close phylogenetic relationship. A major example can be made with IL10: the primer pair amplified S. sciureus but not A. infulatus DNA. When comparing the sequences obtained with S. sciureus DNA with sequences deposited for other species of Aotus, it became evident a major deletion of 21 bp between positions 268–288 in the sequences of all four Aotus species studied (Supplemental Fig. 1). Any primer designed for Saimiri covering this region would obviously not work for Aotus. Additional efforts will be necessary to describe the sequences of the five genes not amplified with the primer pairs used in this work, with the need to design alternative primers and optimize PCR conditions. In addition, several other Saimiri and Aotus genes shall be object of sequencing in order to allow the construction of more complete tools for analysis of immune and other biological responses in these animals. The data presented here show that the approach used is valid and will continue to be pursued. Conflict of interest None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgments This study was supported with funds from the PDTIS Program (Fiocruz), from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), from the DECIT (Ministry of Health) and from Bank BNP Paribas. The authors thank Drs. Leila Mendonça and Constança Brito for granting access to the PDTIS Sequencing and qRT-PCR facilities, respectively. We also thank Drs Carlos Faro (Director of the National Primate Center/SVS/MS), Alexandre da Costa Linhares and Ronaldo Freitas (from the Virology section, Evandro Chagas Institute), Aldo Valente, Marinete Marins Póvoa and Salma Gomes de Oliveira (from the Parasitology section, Evandro Chagas Institute), for granting access to the facilities, animals and equipments decisive for completion of this work, and Silvanira Barbosa for technical assistance at the DNA Polymorphism Laboratory (UFPA). Francisco Acácio Alves received a Doctoral fellowship


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Alves FA