Cutaneous Reactions to Tick Bites and Antigens in Bovines

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Reverse Immunology Screening procedures

• 1 Delayed cutaneous cellular responses to
  antigens delivered as naked DNA in TOPO VR2001
  vector
• Macroscopic erythema and edema
• Histology Basophils, CD3/CD4 mononuclear cells
• Molecular expression of IGF-1, MIP-1alpha, MCP-1
• 2 Th1 responses
• Molecular expression of IFN-?, IP-10
• 3 Antibodies (IgG1 and IgG2, not IgE) and
  recognition of tick salivome with sera from
  naturally immune hosts

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FEATURES OF TOPO VR2001 VECTOR FOR DELIVERY OF 48
ANTIGENS AS DNA FOR IN VIVO SCREENING

• TOPO TA 5 minutes cloning
• Strong Vical CMV promoter
• Signal peptide from tissue plasminogen activator.
  
• makes expressed protein get secreted by
  transfected cells and thus antigen will be
  processed by the class II pathway
• Because of what is known about immune correlates
  of resistance against ticks, we want to favor the
  Class II pathway.
• Developed by Jesus Valenzuela, NIAID-NIH

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Incidence of positive skin reactions in bovines
naturally immune to ticks and inoculated with
naked DNA coding for different salivary antigens.
Antigens were inoculated in duplicates in same
host and some antigens induced opposite
reactions in the same host erratic expression of
protein from naked DNA?
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Further Work - Some Questions

• Erratic responses to Antigens delivered as DNA
• Is protein really expressed by transfected
  cutaneous cells?
• Should we go for recombinant proteins?
• Should we rely on in vitro transfection of DCs
  followed by in vitro readouts?

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Reverse Immunogenomics

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Resistant
Susceptible

• Contrasting phenotypes in bovines
• Is composition of tick saliva affected by S and R
  host immune responses?
• Is this phenomenon accompanied by differential
  expression of genes in Salivary Glands of ticks?
• Does this phenomenon affect choice/performance of
  candidate antigens?

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• Hypothesis Different levels of host anti-tick
  immunity affect expression of genes in salivary
  glands of cattle ticks
• Objectives identify genes of larvae and of
  salivary glands, the expression of which is
  affected by immune responses of susceptible and
  resistant hosts

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Experimental Design
R
Nymphs Males Females
Unfed Larvae (UFL)
Salivary Glands (SG)
S
Nymphs Males Females
mRNA isolation
mRNA isolation
cDNA Library (SMART-Clontech/BD) in ?
TriplEx2 Mass sequencing of ESTs Bioinformatics
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RESULTS General profile of individual cDNA
Libraries
General profile of combined Transcriptomes
RMallSxR 3,342 contigs 7,923 ESTs 312
Contigs SP (9.3) 648 contigs no match
(19.4)
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RESULTS Some clusters with interesting matches
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1 clone female SG e value 5e-004 plancitoxin I
Acanthaster planci starfish (Echinoderma) GRPs
are more abundant in salivary glands of
Dermacentor andersoni females than in those of
Rh. microplus heteroxene tick X monoxene tick?
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RESULTS Statistics of important transcripts for
tick blood meals (?2 test)
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Uninfested skin
Infested skin
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• Unfed male ticks already express IGBPs B and C,
  but not IGBP A (2 clones each from S and from R
  SGs)
• Host response does not affect expression of
  IGBPs, but levels of total IgG and of anti-saliva
  IgG antibodies decrease in S hosts during heavy
  infestations
• - Frequencies of IgG allotypes differ in R and S
  hosts

Kashino S, et al. Exp Parasitol, 2005
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Further Work - Some Questions

• Confirm findings with qRT-PCR
• Significance of differential responses
• Are the responses of R hosts more efficient
  against tick anti-coagulants?
• If S hosts do not make efficient responses
  against tick anti-coagulants, can a vaccine for S
  hosts achieve this?
• Inefficient feeding inhibits gene expression, but
  does reverse immunogenomics identify the correct
  targets?
• ??ltThe products of genes that are affected by
  host responses may be further down a cascade of
  events, the beginning of which contain the bona
  fide targets for a vaccinegt??

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Other Applications of the CAS Bioinformatics Tools

• Count Tables of cDNA libraries of salivary glands
  from females of
• Rhipicephalus microplus
• monoxene bovines short hipostome
• Rhipicephalus sanguineus
• heteroxene dogs - short hipostome
• Amblyomma cajennense
• heteroxene many species of mammals long
  hipostome

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Other Applications of the CAS Bioinformatics Tools
Significant differences, P lt 0.05, chi square
test

• Relative abundance of glycine rich-proteins
  correlates with biology of life cycle and size of
  hipostome

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Universidade de São Paulo Sandra Regina
Maruyama Lucinda Bandão Daniela Moré Elen
Anatriello Antonio Abatepaulo Wanessa
Carvalho Gustavo Rocha Alessandra Franzin Profa.
Dra. Beatriz Rossetti Ferreira Prof. Dr. João
Santana da Silva NIH José Marcos Chaves
Ribeiro Jesus G. Valenzuela Jennifer M. Anderson