High-throughput profiling of the humoral immune responses against thirteen human papillomavirus types by proteome microarrays

Abstract

We have developed microarrays with all eight proteins encoded by 13 different human papillomavirus types associated with anogenital cancer (HPV-16, 18, 31, 33, 35, 45, 53), genital warts (HPV-6, 11), or skin lesions (HPV-1, 2, 4, 5). We analyzed the seroprevalence of antibodies in 546 patients, which had either cervical carcinomas, or precursor lesions, or which were asymptomatic. All patient groups contained sera ranging from high reactivity against multiple HPV proteins to low or no reactivity. Computational analyses showed the E7 proteins of carcinogenic HPV types as significantly more reactive in cancer patients compared to asymptomatic individuals and discriminating between cancer and HSIL or LSIL patients. Antibodies against E4 and E5 had the highest seroprevalence, but did not exhibit differential reactivity relative to pathology. Our study introduces a new approach to future evaluation of the overall antigenicity of HPV proteins and cross-reaction between homologous proteins.

Immunodominant “Asymptomatic” Herpes Simplex Virus 1 and 2 Protein Antigens Identified by Probing Whole-ORFome Microarrays with Serum Antibodies from Seropositive Asymptomatic versus Symptomatic Individuals

“…The construction of HSV-1 and HSV-2 proteome microarrays was described in detail in a previous report (39); proteome microarrays were fabricated, as described previously (19,20,54), by the PCR amplification of coding sequences in genomic DNA, followed by the insertion of amplicons into a T7 expression vector by homologous recombination and expression in coupled in vitro transcriptions-translations (IVTT) prior to printing onto microarrays. Gene sequences for PCR primer design were obtained from the NCBI (accession no.…”

“…HSV-1 strain 17 DNA was supplied as 5 overlapping genomic fragments cloned into cosmids. HSV-2 strain HG52 DNA was supplied as virion-extracted DNA, and primers used for PCR amplification contained 20-bp nucleotides specific for each gene, with an extension of 20 bp complementary to ends of the linear pXT7 vector at the 5= ends (19,20,54). The genome of herpes simplex viruses are CG rich (68% for HSV-1 and 70% for HSV-2).…”

“…HSV-2 strain 333 DNA was prepared from virion-extracted DNA. Open reading frames were amplified and cloned into the T7 vector pXi, as described previously (19,54). Recombinant plasmids were verified by "QC-PCR" (in which the correct size of the insert was verified by PCR) and/or by sequencing.…”

Current Problems and Recent Advances in the Molecular Diagnosis of Genital Human Papillomavirus Infections

“…Serolojik yöntemlerin aktif ve geçirilmiş enfeksiyonları ayırt edememeleri sebebiyle, enfeksiyon tamamen temizlendiği halde antikor testlerinin pozitif olması da bir diğer problemdir. Tüm bu bahsedilen nedenler, serolojik testlerin HPV enfeksiyonlarının tanısında kullanılmasına veya hastalık ilerlemesi için bir prediktif belirteç olarak değerlendirilmesine engel olmaktadır 8,17,24 . Sonuç olarak virusun rutin hücre kültürü ortamlarında üretilememesi, serolojik testlerin yukarıda değinilen sınırlamaları, elektron mikroskopi ve immünohistokimya gibi klasik direkt virolojik tanı yöntemlerinin duyarlılık ve özgüllük eksikliği gibi nedenlerden dolayı, HPV enfeksiyonlarının rutin mikrobiyolojik tanısı için önerilen test formatı temel olarak viral nükleik asitlerin tespitine dayanmaktadır 10,17 .…”

“…Bu yaklaşım, hedef genlerin PCR ile amplifi kasyonu, in-vivo rekombinasyon (klonlama), proteinlerin in-vitro ekspresyonu ve mikroçipler üzerine yazdırılması sonrasında serumdaki antikor yanıtının saptanması prensibine dayanır. Luevano ve arkadaş-ları 24 MALDI-TOF kütle spektrometresi, HPV enfeksiyonlarının tanısı için kullanılmaya baş-layan bir diğer yeni yöntemdir. Üç farklı yöntemin kombine edildiği PCR-RFMP (restriksiyon fragman kütle polimorfi zmi) testinde; PCR amplifi kasyonu, restriksiyon enzimi ile parçalama ve MALDI-TOF kütle spektrometresi ile analiz basamakları bulunmaktadır.…”