Osvaldo J. Lopez

Identification of Neutralizing and Nonneutralizing Epitopes in the Porcine Reproductive and Respiratory Syndrome Virus GP5 Ectodomain

After infection of swine with porcine reproductive and respiratory syndrome virus (PRRSV), there is a rapid rise of PRRSV-specific nonneutralizing antibodies (NNA), while neutralizing antibodies (NA) are detectable not sooner than 3 weeks later. To characterize neutralizing epitopes, we selected phages from a 12-mer phage display library using anti-PRRSV neutralizing monoclonal antibody (MAb) ISU25-C1. In addition, phages carrying peptides recognized by swine antibodies with high seroneutralizing titer were isolated after subtracting from the library those clones binding to swine anti-PRRSV serum with no neutralizing activity. Two epitopes located in the ectodomain of PRRSV GP5 were identified. One of these epitopes, which we named epitope B, was recognized both by neutralizing MAb ISU25-C1 and swine neutralizing serum (NS) but not by swine nonneutralizing serum (NNS), indicating that it is a neutralizing epitope. Epitope B is sequential, conserved among isolates, and not immunodominant. Antibodies directed against it are detected in serum late after infection. In contrast, the other epitope, which we named epitope A, is hypervariable and immunodominant. Antibodies against it appear early after infection with PRRSV. This epitope is recognized by swine NNA but is not recognized by either neutralizing MAb ISU25-C1 or swine NA, indicating that it is not involved in PRRSV neutralization. During infection with PRRSV, epitope A may act as a decoy, eliciting most of the antibodies directed to GP5 and delaying the induction of NA against epitope B for at least 3 weeks. These results are relevant to the design of vaccines against PRRSV.

Protection against Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Infection through Passive Transfer of PRRSV-Neutralizing Antibodies Is Dose Dependent

ABSTRACT Previous work in our laboratory demonstrated that passive transfer of porcine reproductive and respiratory syndrome virus (PRRSV)-neutralizing antibodies (NA) protected pregnant sows against reproductive failure and conferred sterilizing immunity in sows and offspring. We report here on the dose requirement for protection by passive transfer with NA in young weaned pigs. The presence of a 1:8 titer of PRRSV-NA in serum consistently protected pigs against viremia. Nevertheless, their lungs, tonsils, buffy coat cells, and peripheral lymph nodes contained replicating PRRSV similar to the infected control group. Likewise, these animals excreted infectious virus to sentinels similar to the infectivity control animals. In an attempt to reach complete protective immunity equivalent to that previously observed in sows, the pigs were transferred with a higher titer of PRRSV-NA (1:32), and even then apparent sterilizing immunity was attained in only 50% of the animals. In conclusion, the presence of anti-PRRSV-NA in serum with a titer of 1:8 is enough to block viremia but not peripheral tissue seeding and transmission to contact animals. While a relatively low level of NA in blood is capable of conferring sterilizing immunity against PRRSV in sows, the amount of NA necessary to obtain full protection of a young weaned pig would be significantly higher, suggesting that differences exist in the PRRSV pathogenesis between both age groups. In addition, the titer of NA could be a helpful parameter of protection in the assessment of PRRSV vaccines.

Presence of bovine viral diarrhoea virus in lymphoid cell populations of persistently infected cattle.

Cattle infected in utero with bovine viral diarrhoea virus (BVDV) often develop a lifelong persistent infection (PI). During this PI, BVDV infects many cell types including peripheral blood mononuclear cells (PBMNC). To define the lymphoid cell populations in which BVDV persists PBMNC subpopulations were separated using monoclonal antibodies to cell surface markers. Separated cells were analysed by a sensitive PCR assay for BVDV, in conjunction with flow cytometry to identify antigen-containing cells and with viral infectivity assays. The results indicate that BVDV establishes a productive PI in monocytes and T cells bearing the marker BoCD4, BoCD8 or gamma-delta T cell receptor. BVDV was not detected in B cells as a productive nor a latent infection.

Comparison of the immune response elicited by infectious and inactivated foot-and-mouth disease virus in mice

The immune response to foot-and-mouth disease virus (FMDV) elicited by infection or immunization with inactivated virus in adult mice was examined. A model of adoptive transfer of immunocompetent cells was used for this purpose. The results presented here indicate that both short- and long-term secondary immune responses elicited by high doses of inactivated virus are indistinguishable, at the humoral or cellular level, from that observed after infection. The responses to inactivated or infectious virus were both efficiently mediated by B cells. However, immunization with low doses of inactivated virus induced a response which, although effective in aborting infection, was fully dependent on FMDV-specific T cell cooperation. These findings suggest that the different immune responses observed after infection and immunization are mainly the result of the different viral mass presented to the immune system in each case.

Immune Response to Foot-and-Mouth Disease Virus in an Experimental Murine Model II. Basis of Persistent Antibody Reaction

A murine model was used to study the mechanisms involved in the prolonged immune response to live and inactivated foot-and-mouth disease virus (FMDV). The antibody response elicited by the infection persisted throughout the entire life of the animal, while immunization with inactivated virus induced a transient response. The administration of inactivated virus in a water-in-oil emulsion increased antibody titres to values as high as those obtained by infection. There was a high correlation between neutralizing antibody titre and transfer of immunity with primed cells, and the protection afforded against challenge with infectious virus. It appears that the mechanism involved in the induction of prolonged immune memory in infected animals is not due to viral persistence. Nude mice infected with FMDV also evidenced a prolonged immune response, showing marked differences in antibody levels but equal effectiveness against challenge when nu/nu and nu/+ animals were compared. Furthermore, athymic and euthymic littermates were efficient in conferring protection when cells were transferred to irradiated animals. It is concluded that there is an effective, T-cell-independent, prolonged immune memory against FMDV in this murine model, and that the difference in the immune responses to live and inactivated virus is due mainly to differential antigenic processing rather than to a difference in the degree of sensitization of effector cells.

Evaluation of PCR for diagnosis of bovine viral diarrhea virus in tissue homogenates.

Tissue homogenates from 60 specimens submitted to the Veterinary Diagnostic Center were evaluated by polymerase chain reaction (PCR) for detection of bovine viral diarrhea virus (BVDV). Conventional virus isolation procedures showed the specimens contained BVDV. The BVDV RNA was extracted from the homogenates and subjected to a reverse transcription reaction followed by PCR amplification. The PCR product was blotted onto a nylon membrane and hybridized with a 30-base pair oligonucleotide probe labeled with 32P. One set of PCR primers detected BVDV in 46/60 (77%) of the tissue homogenates. An additional set of primers was used to detect 10/11 samples that had escaped detection with the first set of primers. The results indicate that BVDV can be detected by PCR directly out of tissue homogenates generated in a diagnostic setting.

Genotyping of DNA using sequence-specific methyltransferases followed by immunochemical detection.

Abstract Modern molecular genetics relies on the ability to map the positions of genes on chromosomes, relative to known DNA markers. The first such DNA markers described were Restriction Fragment Length Polymorphisms, but any restriction endonuclease used for RFLP mapping is just one member of a restriction-modification pair. For each restriction endonuclease, there is a companion methyltransferase (MTase) that has the same DNA sequence specificity. Therefore, in principle, it should be possible to use MTases rather than restriction enzymes to detect polymorphic sites in DNA. We have used sequence-specific DNA MTases to detect genetic polymorphisms in closely related viral pathogens. If at least one MTase recognition site is present in PCR-amplified DNA, then methyl groups are incorporated; if no MTase site is present, then methyl groups are not incorporated. When several different sequence-specific DNA MTase reactions are carried out, the pattern of methyl incorporation defines a DNA MTase genotype. DNA MTase Genotyping (DMG) can be used to rapidly diagnose heritable or infectious diseases, to immunochemically detect DNA at defined 2 to 8 base pair sites, or to characterize the amplicons by constructing ordered maps.

Does team-based learning improve performance in an infectious diseases course in a preclinical curriculum?

Objectives To examine whether introduction of Team-based Learning (TBL) improves student learning resulting in improved performance on final examination questions and decreased failures in an infectious diseases course. Methods To improve mastery of course content, we designed an intervention, which provided weekly TBL exercises in study years 2 and 3 to review concepts presented during didactic lectures and laboratory exercises. The remaining course structure and content was essentially unchanged. All students taking the course (n=50 in year 1, n=64 in year 2, and n=72 in year 3) participated in this study. Student final examination performance and performance on individual final examination questions were collected and analyzed for changes in response to the study intervention. Results Addition of weekly TBL exercises improved student performance on the course final examination as demonstrated by a statistically significant increase in the distribution of correct answer percentages for questions in common between the final examinations in years 1 and 2 and between years 1 and 3 (t(99) = 3.1454, p<0.05 and t(99) = 4.1268, p<0.01, respectively; Student-Newman-Keuls). There was no statistical difference (t(97) = 0.9814, p> 0.05; Student-Newman-Keuls) in the distribution of correct answer percentages between years two and three. There was also a decrease in final examination failures in years two and three. Conclusions The results suggest that TBL could be used to improve mastery and retention of course content in a preclinical infectious diseases course. Weekly exercises allow students to identify and ameliorate weaknesses in understanding and make adjustments early in the course.

Unusual amino acid usage in the variable regions of mercury-binding antibodies

Monoclonal antibodies (mAb) specific for mercuric ions were isolated from BALB/c mice injected with a mercury‐containing, hapten‐carrier complex. The antibodies reacted by enzyme‐linked immunosorbent assay with bovine serum albumin‐glutathione‐mercuric chloride (BSA‐GSH‐HgCl) but not with BSA‐GSH without mercury. Nucleotide sequences from polymerase chain reaction products encoding six of the antibody heavy‐chain variable regions and seven light‐chain variable regions revealed that all the antibodies contained an unpaired cysteine residue in one hypervariable region, which is unusual for murine antibodies. Mutagenesis of the cysteine to either tyrosine or serine in one of the Hg‐binding antibodies, mAb 4A10, eliminated mercury binding. However, of two influenza‐specific antibodies that contain cysteine residues at the same position as mAb 4A10, one reacted with mercury, although not so strongly as 4A10, whereas the other did not react at all. These results suggested that, in addition to an unpaired cysteine, there are other structural features, not yet identified, that are important for creating an appropriate environment for mercury binding. The antibodies described here could be useful for investigating mechanisms of metal‐protein interactions and for characterizing antibody responses to structurally simple haptens. Proteins 1999;37:429–440. ©1999 Wiley‐Liss, Inc.

Mouse × pig chimeric antibodies expressed in baculovirus retain the same properties of their parent antibodies

The development of hybridoma and recombinant DNA technologies has made it possible to use antibodies against cancer, autoimmune disorders, and infectious diseases in humans. These advances in therapy, as well as immunoprophylaxis, could also make it possible to use these technologies in agricultural species of economic importance such as pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus causing very important economic losses to the industry. Passive transfer of antibodies obtained by biotechnology could be used in the future to complement or replace vaccination against this and other pig pathogens. To this end, we constructed and studied the properties of chimeric mouse × pig anti‐PRRSV antibodies. We cloned the constant regions of gamma‐1 and gamma‐2 heavy chains and the lambda light chain of pig antibodies in frame with the variable regions of heavy and light chains of mouse monoclonal antibody ISU25C1, which has neutralizing activity against PRRSV. The coding regions for chimeric IgG1 and IgG2 were expressed in a baculovirus expression system. Both chimeric antibodies recognized PRRSV in ELISA as well as in a Western‐blot format and, more importantly, were able to neutralize PRRSV in the same fashion as the parent mouse monoclonal antibody ISU25C1. In addition, we show that both pig IgG1 and IgG2 antibodies could bind complement component C1q, with IgG2 being more efficient than IgG1 in binding C1q. Expressing chimeric pig antibodies with protective capabilities offers a new alternative strategy for infectious disease control in domestic pigs.