This article is the result of an initiative between the European Federation of Pharmaceutical Industries Associations (EFPIA) and the European Centre for the Validation of Alternative Methods (ECVAM).Its objectives are to provide the researcher in the safety evaluation laboratory with an up-to-date, easyto-use set of data sheets to aid in the study design process whilst at the same time affording maximum welfare considerations to the experimental animals.Although this article is targeted at researchers in the European Pharmaceutical Industry, it is considered that the principles underpinning the data sets and refinement proposals are equally applicable to all those who use these techniques on animals in their research, whether in research institutes, universities or other sectors of industry. The implications of this article may lead to discussion with regulators, such as those responsible for pharmacopoeial testing.There are numerous publications dealing with the administration of test substances and the removal of blood samples, and many laboratories also have their own 'in-house' guidelines that have been developed by custom and practice over many years. Within European Union Directive 86/609EEC 1 we have an obligation to refine experiments to cause the minimum amount of stress. We hope that this article will provide background data useful to those responsible for protocol design and review.This guide is based on peer-reviewed publications whenever possible, but where this is not possible we have used 'in-house' data and the experience of those on the working party (as well as helpful comments submitted by the industry) for a final opinion. The guide also addresses the continuing need to refine the techniques associated with the administration of substances and the withdrawal of blood, and suggests ways of doing so. Data-sharing between laboratories should be encouraged to avoid duplication of animal work, as well as sharing practical skills concerning animal welfare and scientific problems caused by 'overdosing' in some way or another. The recommendations in this guide refer to the 'normal' animal, and special consideration is needed, for instance, during pregnancy and lactation. Interpretation of studies may be confounded when large volumes are administered or excessive sampling employed, particularly if anaesthetics are used. Copyright The objectives of the Technical Sub group of EFPIA/ ECVAM were as follows:(i) to provide a guide on administration volumes for use in common laboratory species used in toxicity studies required by regulatory authorities; (ii) to provide consensus dosage levels for routine use that represent good practice in terms of animal welfare and practicality; (iii) to produce a guide to dosage levels representing the upper limit of common practice, which leaves scope to make the case for special investigations. Some of these suggested maximum values have been obtained from recent literature, 3,4 but appear high when compared with 'good practice' values. The need for careful attention...

A 20-year-old female hypogammaglobulinemic patient received monotypic Sabin 3 vaccine in 1962. The patient excreted type 3 poliovirus for a period of 637 days without developing any symptoms of poliomyelitis, after which excretion appeared to have ceased spontaneously. The evolution of Sabin 3 throughout the entire period of virus excretion was studied by characterization of seven sequential isolates from the patient. The isolates were analyzed in terms of their antigenic properties, virulence, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 3 vaccine. The isolates followed a main lineage of evolution with a rate of nucleotide substitution that was very similar to that estimated for wild-type poliovirus during person-to-person transmission. There was a delay in the appearance of antigenic variants compared to sequential type 3 isolates from healthy vaccines, which could be one of the possible explanations for the long-term excretion of virus from the patient. The distribution of mutations in the isolates identified regions of the virus possibly involved in adaptation for growth in the human gut and virus persistence. None of the isolates showed a full reversion of the attenuated and temperature-sensitive phenotypes of Sabin 3. Information of this sort will help in the assessment of the risk of spread of virulent polioviruses from long-term excretors and in the design of therapies to stop long-term excretion. This will make an important contribution to the decision-making process on when to stop vaccination once wild poliovirus has been eradicated.

Vaccination with live attenuated simian immunodeficiency virus (SIVmacC8) confers potent, reproducible protection against homologous wild-type virus challenge (SIVmacJ5). The ability of SIVmacC8 to confer resistance to superinfection with an uncloned ex vivo derivative of SIVmac251 (SIVmac32H/L28) was investigated. In naïve, Mauritian-derived cynomolgus macaques (Macaca fascicularis), SIVmac32H/L28 replicated to high peak titres (.10 8 SIV RNA copies ml), persisted at high levels and induced distinctive pathology in lymphoid tissues. In cynomolgus macaques vaccinated with SIVmacC8, no evidence of detectable superinfection was observed in 3/8 vaccinates following challenge 3 or 20 weeks later with SIVmac32H/L28. Analyses after SIVmac32H/L28 challenge revealed a significant reduction in viral RNA (P,0.001) and DNA levels between 20 week vaccinates and challenge controls. Amongst 3 week vaccinates, less potent protection was observed. However, analysis of env from breakthrough virus indicated .99 % sequence similarity with the vaccine virus. Highly sensitive PCR assays that distinguish vaccine and challenge virus stocks demonstrated restimulation of replication of the vaccine virus SIVmacC8 in the face of potent protection against a vigorous, homologous challenge virus. Vaccine-induced antiviral neutralizing antibodies and anti-Nef CD8 + cytotoxic T cell responses did not correlate with the outcome of the challenge. Defining the mechanism of vaccine protection will need to account for the effective control of a genetically closely related challenge virus whilst remaining unable to suppress replication of the pre-existing vaccine virus. The role of innate and intrinsic anti-retroviral immunity in the protection conferred by live attenuated SIV vaccines warrants careful study.

In order to test the hypothesis that CD8 ؉ cytotoxic T lymphocytes mediate protection against acute superinfection, we depleted >99% of CD8 ؉ lymphocytes in live attenuated simian immunodeficiency virus macC8 (SIVmacC8) vaccinees from the onset of vaccination, maintained that depletion for 20 days, and then challenged with pathogenic, wild-type SIVmacJ5. Vaccinees received 5 mg per kg of humanized anti-CD8 monoclonal antibody (MAb) 1 h before inoculation, followed by the same dose again on days 3, 7, 10, 13, and 17. On day 13, peripheral CD8 ؉ T lymphocytes were >99% depleted in three out of four anti-CD8 MAb-treated vaccinees. At this time attenuated SIVmacC8 viral RNA loads in anti-CD8 MAb-treated vaccinees were significantly higher than control vaccinees treated contemporaneously with nonspecific human immunoglobulin. Lymphoid tissue CD8 ؉ T lymphocyte depletion was >99% in three out of four anti-CD8 MAb-treated vaccinees on the day of wild-type SIVmacJ5 challenge. All four control vaccinees and three out of four anti-CD8 MAb-treated vaccinees were protected against detectable superinfection with wild-type SIVmacJ5. Although superinfection with wild-type SIVmacJ5 was detected at postmortem in a single anti-CD8 MAb-treated vaccinee, this did not correlate with the degree of preceding CD8 ؉ T lymphocyte depletion. Clearance of attenuated SIVmacC8 viremia coincided with recovery of normal CD8 ؉ T lymphocyte counts between days 48 and 76. These results support the view that cytotoxic T lymphocytes are important for host-mediated control of SIV primary viremia but do not indicate a central role in protection against acute superinfection conferred by inoculation with live attenuated SIV.

The identification of mechanisms that prevent infection with human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) would facilitate the development of an effective AIDS vaccine. In time-course experiments, protection against detectable superinfection with homologous wild-type SIV was achieved within 21 days of inoculation with live attenuated SIV, prior to the development of detectable anti-SIV humoral immunity. Partial protection against superinfection was achieved within 10 days of inoculation with live attenuated SIV, prior to the development of detectable anti-SIV humoral and cellular immunity. Furthermore, co-inoculation of live attenuated SIV with wild-type SIV resulted in a significant reduction in peak virus loads compared to controls that received wild-type SIV alone. These findings imply that innate immunity or non-immune mechanisms are a significant component of early protection against superinfection conferred by inoculation with live attenuated SIV.

To determine the role that cellular immune responses play in the protection conferred by vaccination with attenuated SIVmac32H (pC8), we have attempted to deplete macaques of their CD8+ cells prior to challenge with wild-type SIVmac32H (pJ5). In two of four pC8-infected macaques, N109 and N112, a transient partial depletion of CD8+ cells by antibody treatment was achieved. On the day of challenge peripheral CD2+CD4-CD8+ cell counts were reduced by 92 and 95%, respectively, in animals N109 and N112 and their lymph nodes revealed a 46 and 58% reduction, respectively, in CD2+CD4-CD8+ cells. Two other pC8-immunized macaques, N110 and N111, treated in the same way, did not show significant depletion of CD8+ cells. None of these four pC8-immunized animals became infected when challenged with 50 MID50 of pJ5. Treatment of a further four pC8-infected and protected macaques and two naive control animals with Campath-1H antibody successfully depleted peripheral CD3+ cell counts by >99% in all treated animals. Campath-1H depletion resulted in enhanced, longer lasting lymphoid depletion. Yet subsequent challenge with 20 MID50 of pJ5 still failed to infect the pC8-immunized animals. All eight of the naive controls, including two Campath-1H-treated animals, became infected following challenge. In summary, partial depletion of circulating CD8+ cells or total lymphocytes prior to challenge failed to abrogate the protection conferred by vaccination with pC8.

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The distribution of virus-infected cells in cynomolgus macaques was determined at 4, 7, 14 and 28 days following intravenous challenge with 1000 TCID 50 of the wild-type simian immunodeficiency virus SIVmacJ5 (stock J5C). At each time-point, pairs of macaques were killed humanely and the presence of SIV was determined and quantified in blood, spleen, peripheral and mesenteric lymph nodes, thymus, lung and ileum by virus co-cultivation with C8166 cells, by quantitative DNA PCR or by in situ hybridization (ISH). At day 4 post-infection (p.i.), detection of the virus was sporadic. By day 7 p.i., however, significant SIV loads were detected in the blood and lymphoid tissues by DNA PCR and virus co-cultivation. Large numbers of cells expressing SIV RNA were detected in mesenteric lymph nodes by ISH and significantly fewer (P 0n05) in the spleen. Significant numbers of ISH-positive cells were also observed in sections of ileum. By day 14 p.i., the distribution of SIV was more even in all lymphoid tissues analysed. By day 28, most of the tissues were negative by ISH, but all remained positive by virus isolation and DNA PCR. Immunolabelling of sections of mesenteric lymph node with monoclonal antibodies specific for SIV envelope and Nef largely confirmed the observations from ISH. These results indicate that, even following intravenous challenge, a major site of the initial replication of SIV is gut-associated lymphoid tissue. Vaccines that induce protection at this site may therefore be superior, even against parenteral challenge.