Advances in the treatment of cytomegalovirus.

BACKGROUND: Human cytomegalovirus (HCMV) is a threat to immunologically weak patients. HCMV cannot yet be eliminated with a vaccine, despite recent advances. SOURCES OF DATA: Sources of data are recently published research papers and reviews about HCMV treatments. AREAS OF AGREEMENT: Current antivirals target the UL54 DNA polymerase and are limited by nephrotoxicity and viral resistance. Promisingly, letermovir targets the HCMV terminase complex and has been recently approved by the FDA and EMA. AREAS OF CONTROVERSY: Should we screen newborns for HCMV, and use antivirals to treat sensorineural hearing loss after congenital HCMV infection? GROWING POINTS: Growing points are developing drugs against latently infected cells. In addition to small molecule inhibitors, a chemokine-based fusion toxin protein, F49A-FTP, has shown promise in killing both lytically and latently infected cells. AREAS TIMELY FOR DEVELOPING RESEARCH: We need to understand what immune responses are required to control HCMV, and how best to raise these immune responses with a vaccine.

LPS promotes a monocyte phenotype permissive for human cytomegalovirus immediate-early gene expression upon infection but not reactivation from latency.

Human cytomegalovirus (HCMV) infection of myeloid cells is closely linked with the differentiation status of the cell. Haematopoietic progenitors and CD14+ monocytes are usually non-permissive for lytic gene expression which can lead to the establishment of latent infections. In contrast, differentiation to macrophage or dendritic cell (DC) phenotypes promotes viral reactivation or renders them permissive for lytic infection. The observation that high doses of Lipopolysaccharide (LPS) drove rapid monocyte differentiation in mice led us to investigate the response of human monocytes to HCMV following LPS stimulation in vitro. Here we report that LPS triggers a monocyte phenotype permissiveness for lytic infection directly correlating with LPS concentration. In contrast, addition of LPS directly to latently infected monocytes was not sufficient to trigger viral reactivation which is likely linked with the failure of the monocytes to differentiate to a DC phenotype. Interestingly, we observe that this effect on lytic infection of monocytes is transient, appears to be dependent on COX-2 activation and does not result in a full productive infection. Thus LPS stimulated monocytes are partially permissive lytic gene expression but did not have long term impact on monocyte identity regarding their differentiation and susceptibility for the full lytic cycle of HCMV.

Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells.

Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle. We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation. In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells. This approach of transiently inducing viral lytic gene expression by HDAC inhibition, in otherwise latently infected cells, offers a window of opportunity to target and purge the latent myeloid cell reservoir by making these normally immunologically undetectable cells visible to pre-existing host immune responses to viral lytic antigens.

Diverse specificities, phenotypes, and antiviral activities of cytomegalovirus-specific CD8+ T cells.

UNLABELLED: CD8(+) T cells specific for pp65, IE1, and IE2 are present at high frequencies in human cytomegalovirus (HCMV)-seropositive individuals, and these have been shown to have phenotypes associated with terminal differentiation, as well as both cytokine and proliferative dysfunctions, especially in the elderly. However, more recently, T cell responses to many other HCMV proteins have been described, but little is known about their phenotypes and functions. Consequently, in this study, we chose to determine the diversity of HCMV-specific CD8(+) T cell responses to the products of 11 HCMV open reading frames (ORFs) in a cohort of donors aged 20 to 80 years old as well as the ability of the T cells to secrete gamma interferon (IFN-γ). Finally, we also tested their functional antiviral capacity using a novel viral dissemination assay. We identified substantial CD8(+) T cell responses by IFN-γ enzyme-linked immunospot (ELISPOT) assays to all 11 of these HCMV proteins, and across the cohort, individuals displayed a range of responses, from tightly focused to highly diverse, which were stable over time. CD8(+) T cell responses to the HCMV ORFs were highly differentiated and predominantly CD45RA(+), CD57(+), and CD28(-), across the cohort. These highly differentiated cells had the ability to inhibit viral spread even following direct ex vivo isolation. Taken together, our data argue that HCMV-specific CD8(+) T cells have effective antiviral activity irrespective of the viral protein recognized across the whole cohort and despite viral immune evasion. IMPORTANCE: Human cytomegalovirus (HCMV) is normally carried without clinical symptoms and is widely prevalent in the population; however, it often causes severe clinical disease in individuals with compromised immune responses. HCMV is never cleared after primary infection but persists in the host for life. In HCMV carriers, the immune response to HCMV includes large numbers of virus-specific immune cells, and the virus has evolved many mechanisms to evade the immune response. While this immune response seems to protect healthy people from subsequent disease, the virus is never eliminated. It has been suggested that this continuous surveillance by the immune system may have deleterious effects in later life. The study presented in this paper examined immune responses from a cohort of donors and shows that these immune cells are effective at controlling the virus and can overcome the virus' lytic cycle immune evasion mechanisms.

Effects of Chlamydia trachomatis infection on the expression of natural killer (NK) cell ligands and susceptibility to NK cell lysis.

Natural killer (NK) cells are an important component of the immediate immune response to infections, including infection by intracellular bacteria. We have investigated recognition of Chlamydia trachomatis (CT) by NK cells and show that these cells are activated to produce interferon (IFN)-gamma when peripheral blood mononuclear cells (PBMC) are stimulated with CT organisms. Furthermore, infection of epithelial cell lines with CT renders them susceptible to lysis by human NK cells. Susceptibility was observed 18-24 h following infection and required protein synthesis by the infecting chlamydiae, but not by the host cell; heat or UV inactivated chlamydiae did not induce susceptibility to NK cell lysis. CT infection was also shown to decrease the expression of classical and non-classical major histocompatibility complex (MHC) molecules on infected cells, thus allowing recognition by NK cells when combined with an activating signal. A candidate activating signal is MICA/B, which was shown to be expressed constitutively on epithelial cells.

Vaccines against persistent DNA virus infections.

Persistent viruses present some particular problems for vaccine design. As for acute non-persistent viruses, the prime goal of a vaccine should be to prevent primary infection. Vaccines might also be used to modify the course of established persistent virus infections - so-called postinfective immunisation. This chapter deals with selected persistent DNA viruses, in particular the human herpes viruses.

Latency and reactivation of human cytomegalovirus.

The sequence analysis of herpesviruses suggests they have been evolving with their individual vertebrate hosts for millions of years, and their divergence parallels that of the hosts they infect. Given this time they have been learning to live with their individual hosts, it is not surprising that they have become extremely well adapted to doing so without causing much in the way of obvious disease. A key feature of their strategy for persisting in the host is the ability of all herpesviruses to establish latent infection-a state in which no, or only a very limited set of, viral genes are expressed in cells in which viral DNA persists. The alpha herpesviruses (herpes simplex and varicella zoster virus) establish latency in neuronal cells in sensory ganglia: these are long lived non-dividing cells and the alpha herpesviruses persist in these with expression of only the latency associated transcripts-although the function of these RNA transcripts remains incompletely understood. The principal gamma herpesvirus of humans, Epstein Barr virus (EBV), is latent mainly in B lymphocytes: EBV persistence in B cells is associated with expression of a limited set of viral genes encoding functions necessary for the maintenance of the episomal viral DNA as B cells divide.The mechanism by which the principal beta herpesvirus of humans-human cytomegalovirus (HCMV) persists, is also incompletely understood and the subject of this review. Understanding how HCMV persists has clinical relevance in that its transmission to seronegative recipients might be more easily prevented, and the mechanisms by which it produces disease in the neonate and immunocompromised hosts more easily understood, if we knew more about the cells in which the virus is latent and the way in which it reactivates.

Functional heterogeneity and high frequencies of cytomegalovirus-specific CD8(+) T lymphocytes in healthy seropositive donors.

Human cytomegalovirus (HCMV) infection is largely asymptomatic in the immunocompetent host, but remains a major cause of morbidity in immunosuppressed individuals. Using the recently described technique of staining antigen-specific CD8(+) T cells with peptide-HLA tetrameric complexes, we have demonstrated high levels of antigen-specific cells specific for HCMV peptides and show that this may exceed 4% of CD8(+) T cells in immunocompetent donors. Moreover, by staining with tetramers in combination with antibodies to cell surface markers and intracellular cytokines, we demonstrate functional heterogeneity of HCMV-specific populations. A substantial proportion of these are effector cytotoxic T lymphocytes, as demonstrated by their ability to lyse peptide-pulsed targets in "fresh" killing assays. These data suggest that the immune response to HCMV is periodically boosted by a low level of HCMV replication and that sustained immunological surveillance contributes to the maintenance of host-pathogen homeostasis. These observations should improve our understanding of the immunobiology of persistent viral infection.

Status of Cytomegalovirus Prevention and Treatment in 2000.

Cytomegalovirus (CMV) infection continues to be a problem in selected populations following hematopoietic stem cell transplantation (SCT). Although there have been no new antiviral agents for management of this infection in recent years, the methods for using the existing agents have improved with newer assays for detection of virus. In addition, our understanding of immunity to CMV has undergone considerable expansion. This paper will address these new aspects relating to CMV infection in the setting of SCT. In Section I Dr. Zaia reviews the pathogenesis of CMV and the current epidemiology of CMV disease following marrow or blood allo-SCT with emphasis on late-onset disease. The current lab tests available for preemptive management are summarized including the role for conventional shell vial cultures, and a comparison of the CMV antigenemia assay with the new nucleic acid-based assays, including the hybrid capture assay, the NASBA assay, and "real-time" PCR assays. Use of antiviral agents with these tests in the preemptive management of CMV infection is discussed. Ultimately, what is necessary is restoration of adequate CMV immunity, and that requires understanding the basics of the CMV-specific immune response. In Section II, Dr. Sissons traces the evolution of the CTL response from primary infection into memory and reviews recent advances in the understanding of cytotoxic T cell based immunity to CMV, based on the use of T cell clonotypic analysis and markers of T cell memory and activation, with conventional CTL functional assays. In Section III Dr. Riddell presents approaches to correction of the problem of CMV pathogenesis, namely direct restoration of the CMV-specific cellular immune deficiency. Attempts at passive therapies will be reviewed with the focus on current problems and approaches to these problems. In Section IV, Dr. Diamond presents work on the identification of multiple HLA-allele specific cytotoxic T cell epitopes specific for CMV-pp65 and - pp150. Specific epitopes are recognized by CMV-seropositive individuals including healthy donors, SCT recipients, and AIDS patients, indicating their potential usefulness as vaccines. One of these epitopes is recognized by most individuals who express the HLA A(*)0201 Class I allele. Pre-clinical evaluation in HLA2.1 transgenic mice of vaccine structures utilizing this epitope, and alternative delivery systems are described. Possible methods for vaccination of donor and/or recipient of a SCT as well as their limitations, utilizing synthetic or viral vaccines, are discusseed.

Analysis of cytotoxic T-cell responses to human cytomegalovirus infection.

Inferential evidence from studies of immunosuppressed humans indicates the control of disease due to human cytomegalovirus (HCMV) in the persistently infected host (virus carrier) depends on the cellular immune response: cytotoxic T lymphocytes (CTL) appear to be a particularly important component of this response (1,2).

Large clonal expansions of human virus-specific memory cytotoxic T lymphocytes within the CD57+ CD28- CD8+ T-cell population.

The proportion of human peripheral blood CD8+ T cells that are CD57+ CD28- is low at birth but increases with age and in individuals infected with human cytomegalovirus (HCMV) or human immunodeficiency virus (HIV). These CD57+ CD28- CD8+ T cells contain large oligoclonal T-cell expansions whose antigen specificity is unknown. We identified clonal expansions of virus-specific memory cytotoxic T-lymphocyte precursors (CTLp) in both healthy carriers of HCMV and in asymptomatic HIV-infected subjects. In each subject, from the T-cell receptor (TCR) beta-chain hypervariable sequence of each immunodominant CTL clone, we designed complementary oligonucleotide probes to quantify the size and phenotypic segregation of individual virus-specific CTL clones in highly purified populations of peripheral blood CD8+ T cells. We found large clonal expansions of virus-specific CTL clonotypes in CD57+ CD28- CD8+ T cells. Using limiting dilution analysis, we found functional peptide-specific CTLp at high frequency in CD57+ CD28- cells. Thus, memory CTL specific for persistent viruses account for many oligoclonal expansions within CD57+ CD28- CD8+ T cells.

Human CD28-CD8+ T cells contain greatly expanded functional virus-specific memory CTL clones.

At birth, almost all human peripheral blood CD8+ T cells express the costimulatory molecule CD28. With increasing age, the proportion of CD8+ T cells that lack CD28 increases. Because the Ag specificity of CD28-CD8+ T cells has not previously been defined, we studied the contribution of CD28-CD8+ T cells to the memory CD8+ CTL response against two human persistent viruses, human CMV (HCMV) and HIV. From PBMC of healthy virus carriers we generated multiple independent CTL clones specific for defined viral peptides and sequenced their TCR beta-chains. We designed clonotypic oligonucleotides complementary to each beta-chain hypervariable sequence and quantified the size of individual immunodominant CTL clones in PBMC. Some individual CTL clones were very large, comprising up to 3.1% of all CD8+ T cells in PBMC, and were generally maintained at a stable level for months. Individual virus-specific CTL clones were consistently more abundant in purified CD28- cells than in the CD8+ population as a whole. Because CD28-CD8+ cells as a population have been reported to proliferate poorly in response to mitogen, we studied the function of these virus-specific CD28- CTL clones by quantifying the frequency of peptide-specific CTL precursors using limiting dilution analysis. CD28-CD8+ T cells contained high frequencies of functional memory CTL precursors specific for peptides of HCMV or HIV, generally higher than in the CD8+ T cell population as a whole. We conclude that in asymptomatic HCMV and HIV infection, human CD28-CD8+ T cells contain high frequencies of functional virus-specific memory CTL clones.

Human virus-specific CD8+ CTL clones revert from CD45ROhigh to CD45RAhigh in vivo: CD45RAhighCD8+ T cells comprise both naive and memory cells.

It has been generally believed that human CD8+ memory cells are principally found within the CD45ROhigh population. There are high frequencies of CD8+ memory CTL specific for the human CMV tegument phosphoprotein pp65 in PBMC of long-term virus carriers; the large population of memory CTL specific for a given pp65 peptide contains individual CTL clones that have greatly expanded. In this study, we found high frequencies of pp65 peptide-specific memory CTL precursors in the CD45ROhighCD45RA- population, but also appreciable frequencies in the CD45RAhigh subpopulation. Because the majority of CD8+ T cells in PBMC are CD45RAhigh, more of the total pp65-specific memory CTL pool is within the CD45RAhigh than in the CD45ROhigh compartment. Using clonotypic oligonucleotide probes to quantify the size of individual pp65-specific CTL clones in vivo, we found the CD45RAhigh population contributed 6- to 10-fold more than the CD45ROhigh population to the total virus-specific clone size in CD8+ cells. During primary CMV infection, an individual virus-specific CTL clone was initially CD45ROhigh, but after resolution of infection this clone was detected in both the CD45ROhigh and the CD45RAhigh populations. We conclude that CD45RA+ human CD8+ T cells do not solely comprise naive cells, but contain a very significant proportion of memory cells, which can revert from the CD45ROhigh to CD45RAhigh phenotype in vivo.

The memory cytotoxic T-lymphocyte (CTL) response to human cytomegalovirus infection contains individual peptide-specific CTL clones that have undergone extensive expansion in vivo.

Human cytomegalovirus (HCMV)-specific CD8(+) cytotoxic T lymphocytes (CTL) appear to play an important role in the control of virus replication and in protection against HCMV-related disease. We have previously reported high frequencies of memory CTL precursors (CTLp) specific to the HCMV tegument protein pp65 in the peripheral blood of healthy virus carriers. In some individuals, the CTL response to this protein is focused on only a single epitope, whereas in other virus carriers CTL recognized multiple epitopes which we identified by using synthetic peptides. We have analyzed the clonal composition of the memory CTL response to four of these pp65 epitopes by sequencing the T-cell receptors (TCR) of multiple independently derived epitope-specific CTL clones, which were derived by formal single-cell cloning or from clonal CTL microcultures. In all cases, we have observed a high degree of clonal focusing: the majority of CTL clones specific to a defined pp65 peptide from any one virus carrier use only one or two different TCRs at the level of the nucleotide sequence. Among virus carriers who have the same major histocompatibility complex (MHC) class I allele, we observed that CTL from different donors that recognize the same peptide-MHC complex often used the same Vbeta segment, although other TCR gene segments and CDR3 length were not in general conserved. We have also examined the clonal composition of CTL specific to pp65 peptides in asymptomatic human immunodeficiency virus-infected individuals. We have observed a similarly focused peptide-specific CTL response. Thus, the large population of circulating HCMV peptide-specific memory CTLp in virus carriers in fact contains individual CTL clones that have undergone extensive clonal expansion in vivo.

Reversal by desferrioxamine of tau protein aggregates following two days of treatment in aluminum-induced neurofibrillary degeneration in rabbit: implications for clinical trials in Alzheimer's disease.

A clinical trial in patients with Alzheimer's disease has indicated that frequent intramuscular (i.m.) treatment with desferrioxamine (DFO) slows progression of the disease. Confirmatory trials have not been carried out, partly because of the rigors of twice daily intramuscular injections over a period of 2 years, even though the initial report gave promising results. The aim of the present study was to determine an optimal DFO treatment protocol in an animal model exhibiting Alzheimer's-like intraneuronal protein aggregates, previously shown to be partially reversed by such treatment. New Zealand white rabbits were injected intracisternally with either aluminum (Al) maltolate or with saline on day 0. Intramuscular injections of DFO were given to selected rabbits for 2 days prior to sacrifice on days 4, 6 or 8. Bielschowsky's silver impregnation demonstrated widespread neurofibrillary degeneration (NFD) in neuronal cell bodies and neurites of brain and spinal cord from Al-treated rabbits. Monoclonal antibodies Tau-2, AT8, PHF-1 and Alz-50, all of which characteristically stain neurofibrillary tangles associated with Alzheimer's disease, strongly labeled the Al-induced NFD. The number of positive neurons and staining intensities were much less in rabbits treated with Al and subsequently with DFO, than in animals only given Al. Control rabbit receiving intracisternal saline were negative for NFD. The results of quantitative immunohistochemistry using image analysis confirmed that immunostaining densities with all tau mAbs were higher in Al-treated than in Al-DFO-treated or in saline-treated controls. Furthermore, it appears that hyperphosphorylation of tau does not make this protein resistant to degradation once Al has been removed by DFO treatment. The effectiveness of only two days of DFO treatment in reversing Al-induced neurofibrillary degeneration suggests that further clinical trials of DFO for treatment of Alzheimer's disease should be attempted using much less frequent administration of DFO than in the initial study (Crapper McLachlan et al., 1991).

Neurofibrillary lesions in experimental aluminum-induced encephalopathy and Alzheimer's disease share immunoreactivity for amyloid precursor protein, A beta, alpha 1-antichymotrypsin and ubiquitin-protein conjugates.

Neurofibrillary tangles of Alzheimer's disease contain predominantly tau protein and to a lesser degree amyloid precursor protein (APP), A beta protein, alpha 1-antichymotrypsin (ACT) and ubiquitin. Previously we have demonstrated the presence of phosphorylated tau and neurofilament proteins in neurofibrillary degeneration (NFD) induced by aluminum (Al) maltolate in rabbits [Savory et al., Brain Res. 669 (1995) 325-329; Savory et al., Brain Res. 707 (1996) 272-281]. Using the same animal system we have now detected APP, A beta, ACT and ubiquitin-like immunoreactivities in NFD-bearing neurons, often colocalizing in the NFD. Diffuse cytoplasmic staining for APP, A beta and ubiquitin was also present in neurons without NFD from Al maltolate-treated rabbits. This study provides additional support for immunochemical similarities between Al-induced NFD in rabbits and the neurofibrillary tangles in human subjects with Alzheimer's disease.

The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL.

Cytotoxic T lymphocytes (CTL) appear to play an important role in the control of human cytomegalovirus (HCMV) in the normal virus carrier: previous studies have identified peripheral blood CD8+ CTL specific for the HCMV major immediate-early gene product (IE1) and more recently, by bulk culture and cloning techniques, have identified CTL specific for a structural gene product, the lower matrix protein pp65. In order to determine the relative contributions of CTL which recognize the HCMV proteins IE1, pp65, and glycoprotein B (gB) to the total HCMV-specific CTL response, we have used a limiting-dilution analysis system to quantify HCMV-specific CTL precursors with different specificities, allowing the antigenic specificity of multiple short-term CTL clones to be assessed, in a group of six healthy seropositive donors. All donors showed high frequencies of HCMV-specific major histocompatibility complex-restricted CTL precursors. There was a very high frequency of CTL specific for pp65 (lower matrix protein); IE1-specific CTL were also detectable at lower frequencies in three of five donors, while CTL directed to gB were undetectable. A pp65 gene deletion mutant of HCMV was then used to estimate the contribution of pp65-specific CTL to the total HCMV-specific CTL response; this showed that between 70 and 90% of all CTL recognizing HCMV-infected cells were pp65 specific. Analysis of the peptide specificity of pp65-specific CTL showed that some donors have a highly focused response recognizing a single peptide; the T-cell receptor Vbeta gene usage in these two donors was shown to be remarkably restricted, with over half of the responding CD8+ T cells utilizing a single Vbeta gene rearrangement. Other subjects recognized multiple pp65 peptides: nine new pp65 CTL peptide epitopes were defined, and for five of these the HLA-presenting allele has been identified. All four of the HLA A2 donors tested in this study recognized the same peptide. This apparent domination of the CTL response to HCMV during persistent infection by a single structural protein, irrespective of major histocompatibility complex haplotype, is not clearly described for other persistent virus infections, and the mechanism requires further investigation.

Quantitative image analysis of temporal changes in tau and neurofilament proteins during the course of acute experimental neurofibrillary degeneration; non-phosphorylated epitopes precede phosphorylation.

Perturbation of the neuronal cytoskeleton represents an integral feature of neurofibrillary tangles which are characteristic neuropathological findings seen in Alzheimer's disease. Microtubule associated protein tau (tau) is considered to be the major component of these lesions although neurofilament proteins also are present. The present study explores the formation of intraneuronal tau and neurofilament protein aggregates using intracisternal administration of aluminum maltolate to rabbits. The time course of the formation of these aggregates and subsequent phosphorylation have been investigated by immunohistochemical methods using a panel of monoclonal antibodies, with quantitation of the staining by image analysis. Neurofilament proteins begin to aggregate by day 1 following aluminum maltolate injection on day 0. Increases in non-phosphorylated neurofilament proteins are observed first, with phosphorylated epitopes being recognized by day 3. Tau follows a similar pattern in that non-phosphorylated epitopes appear to precede phosphorylation. The monoclonal antibody Alz-50 which recognizes a phosphorylation-independent epitope of tau in Alzheimer's disease paired helical filaments, demonstrates positivity in the aluminum maltolate-treated rabbits by day 3. Other tau monoclonal antibodies which recognize phosphorylated tau in paired helical filaments (AT8 and PHF-1) show positive immunostaining on days 6-8. These results indicate that intraneuronal aggregation of cytoskeletal proteins can be initiated by factors other than phosphorylation. However, phosphorylation occurring as a secondary event probably contributes to stabilization of the aggregates.

Passage of Japanese encephalitis virus in HeLa cells results in attenuation of virulence in mice.

Of four wild-type strains (Nakayama-original, SA14, 826309 and Beijing-1) of Japanese encephalitis (JE) virus that were passaged six times in HeLa cells (HeLa p6), two (Nakayama-original and 826309) became attenuated for mice. In the case of strain Nakayama-original, the virulence for mice was markedly reduced and attenuation was retained on passage in primary chicken embryo fibroblast, LLC-MK2 and C6/36 cells. The binding of non-HeLa-passaged Nakayama virus to mouse brain membrane receptor preparations could be differentiated from binding by Nakayama HeLa p6 virus, suggesting that the envelope (E) protein is involved in the attenuated phenotype. Both of the attenuated viruses can be distinguished from the virulent non-HeLa-passaged parental viruses by examination with E protein reactive vaccine and wild-type-specific monoclonal antibodies (MAbs). The vaccine-specific MAb V23, which is only reactive with the SA14 series of live vaccine viruses, recognized the HeLa cell-attenuated Nakayama-original and 826309 viruses, whereas two wild-type-specific MAbs (MAbs K13 and K39) lost reactivity. Comparison of the nucleotide sequences of the structural protein genes of the 826309 and Nakayama-original virulent parent and attenuated HeLa p6 viruses revealed that the viruses differed by 37 and 46 nucleotides coding for eight and nine amino acid mutations, respectively. However, other than one amino acid in the E protein, the membrane and E protein amino acid sequences of the two attenuated HeLa p6 viruses were identical.