Multiple enzyme activities of flavivirus proteins.

Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified -200 compounds having > or = 50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase activities. The NTPase and the 5'-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNA-dependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3' UTR and the RKRK motif in viral replication.

Flow cytometric detection of intracellular cytokines in peripheral blood of HIV-1 infected Thai children.

The objective of this study was to determine changes in Th1/Th2 cytokine production at the cellular level which occur during the progression of HIV-1 subtype E infection in Thai children born to HIV-1 subtype E infected mothers. Mitogen stimulated whole blood cultures from 12 uninfected and 27 HIV-1 subtype E infected Thai children were stained intracellularly with fluorescein labelled monoclonal antibodies against Interleukin (IL)-2 and IFN-gamma (Th1 cytokines) and IL-4 (Th2 cytokine). Additionally, co-staining of CD4+ and CD8+ T cells was performed. Results were analyzed by two and three color flow cytometry. The percentage of IFN-gamma expressing cells in CD4+ T cells was increased in HIV-1 subtype E infected Thai children with mild and moderate immunosuppression (Immunological categories 1 + 2, Centers for Diseases Control and Prevention (CDC) staging system, 1994). The percentages of IFN-gamma expression was continuously enhanced accompanied by remaining preserved in the proportion of IL-2 producing T cells in HIV-1 subtype E infected Thai children with severe immunosuppression (Immunological category 3, CDC staging system, 1994). The percentages of IFN-gamma expression was continuously augmented whereas the proportion of IL-2 producing T cells remained unchanged in HIV-1 subtype E infected Thai Children with severe immunosuppression (immunological category 3, CDC staging system, 1994). The percentage of Th2 cytokine producing cells within the CD4+ ad CD8+ T cells increased in HIV-1 subtype E infected individuals and showed a significant difference in HIV-1 subtype E infected Thai children with AIDS compared with uninfected infants. These results suggest that in vertically acquired HIV-1 infection with severe immunosuppression, the percentages of IL-2 producing CD4+ T cell was consistent but the percentages of IL-4 and IFN-gamma producing cell were increased. Similar results were found for CD8+ T cells in which IL-4 producing cells were increased in conjunction with a remaining in the number of IL-2 producing cells in HIV-1 subtype E infected Thai children. Thus, changes in the Th1 and Th2 cytokine pattern during HIV-1 infection may contribute to the prognosis of HIV disease in children.

A radical form of nitric oxide suppresses RNA synthesis of rabies virus.

Hydrophobia is an incurable disease of the central nervous system. Therefore, every mode of the immune response is important to inhibit and clear infection. Innate immunity such as nitric oxide is significantly upregulated during rabies virus infection in vivo. In this report, the possible role of nitric oxide in inhibition of rabies virus replication was studied. Rabies virus infected neuroblastoma cells were treated with nitric oxide generated from SNP or SNP in the presence of ascorbate. SNP-ascorbate generates mainly NO* in culture medium while NO(+) is the major product of SNP alone. Treatment with SNP-ascorbate resulted in delay and suppression of infectious viral particle production. In contrast, treatment with SNP alone did not interfere with multiplication of this virus. The mechanism of inhibition by NO was at the level of gene expression, which was demonstrated by reduction in the level of N, G and L gene expression. The effect of SNP-ascorbate generated NO on rabies virus protein synthesis was also investigated. Synthesis of N protein in the presence of NO was suppressed which correlated to down regulation of N gene expression. We hypothesize that one of the roles of NO in the central nervous system during rabies virus infection is to limit viral dissemination by down-regulating rabies virus production through transcription inhibition.

TNF-alpha induces caspase 3 (CPP 32) dependent apoptosis in human cholangiocarcinoma cell line.

Cholangiocarcinoma (CCA), a malignant tumor derived from bile duct epithelium, occurs with a higher incidence in tropical countires especially in some areas of Southeast Asian countries such as Thailand. This tumor is relatively resistant to chemotherapy. In this study, molecular mechanism of killing of this tumor by TNF-alpha was investigated. Human cholangiocarcinoma cell line (HuCCA-1) was developed and used as a model for treatment. Activation of HuCCA-1 with TNF-alpha in the present of actinomycin D (1 microg/ml) caused death of the tumor cells. Western blotting analysis of the cells extracted demonstrated the cleavage of poly (ADP-ribose) polymerase (PARP) within 6-8 hours following TNF-alpha treatment indicating apoptotic death. The cleavage of PARP was inhibited when the cell line was pretreated with peptide inhibitor, Ac-DEVD-CHO, suggesting that apoptosis induced by TNF-alpha of this cell line involves activation of caspase II subfamily. The procaspase 3 (proCPP-32), one of the caspase group II subfamily was degraded after the HuCCA- I cell line was treated with TNF-alpha. Furthermore, Gelsolin, an 83 kDa protein which is identified as caspase 3 substrate, was cleaved to 43 kDa fragments after the cells were treated with TNF-alpha. These results indicate that apoptosis of human cholangiocarcinoma cell line as induced by TNF-alpha treatment is mediated through caspase 3.

Binding of tumour necrosis factor-alpha (TNF-alpha) to TNF-RI induces caspase(s)-dependent apoptosis in human cholangiocarcinoma cell lines.

Cholangiocarcinoma (CCA), a tumour of the bile duct epithelium, occurs with a higher incidence in South-east Asian countries than in Europe and North America. The prognosis is poor, due to the unavailability of early diagnosis and the tumours being relatively resistant to chemotherapy. In the present study one of the fatal routes of this tumour was studied. This death was stimulated by TNF-alpha. TNF-alpha at a concentration of 760 pg/ml and 100 pg/ml in the presence of 1 microgram/ml actinomycin D induced 50% cell death of the two established human cholangiocarcinoma cell lines HuCCA-1 and HuCCA-INu, respectively. Preincubation of both cell lines with MoAb to TNF-RI or TNF-RII before TNF-alpha treatment showed that only the MoAb specific to TNF-RI inhibited death. The death of these two cell lines was proved to be apoptosis. Western blot analysis of extracts from both cell lines demonstrated a cleavage of poly (ADP-ribose) polymerase (PARP) within 6-8 h following TNF-alpha treatment. The degradation of PARP was prevented by a MoAb to TNF-RI indicating that the TNF-RI but not TNF-RII was involved in TNF-induced apoptosis in these two human cholangiocarcinoma cell lines. Moreover, peptide inhibitor for caspase II subfamily, Ac-DEVD-CHO, reduced the cytolysis of TNF-alpha-treated cholangiocarcinoma cells. The inhibitor also prevented degradation of PARP. These results indicate that the interaction between TNF-alpha and TNF-RI alone generated a sufficient signal to activate a caspase II subfamily-dependent apoptosis in human cholangiocarcinoma cell lines.

Rabies virus replication induces Bax-related, caspase dependent apoptosis in mouse neuroblastoma cells.

Rabies virus has been shown to induce apoptosis in infected cells, but the intracellular pathway of cell killing is unknown. In this report, we show that rabies virus infected mouse neuroblastoma cells underwent chromatin condensation and DNA fragmentation within 48 h post-infection. An increased level of the apoptotic enhancer, Bax, was detected within 24 h after infection. In contrast to Bax, the production of the apoptotic antagonist, Bcl-2, remained unchanged. Shortly after detection of Bax, caspase 1 (ICE) was upregulated. Reduction of DNA fragmentation in rabies virus infected cultures pretreated with YVAD and DEVD suggested that more than one subfamily of caspase functioned in the death process. Significant degradation of the DNA repair enzyme, poly ADP-ribose polymerase (PARP), was revealed after caspase upregulation. This study showed that replication of rabies viruses in mouse neuroblastoma cells induced the Bax-related death program leading to destruction of the DNA repair system probably by caspase activity.

Apoptosis induction in brain during the fixed strain of rabies virus infection correlates with onset and severity of illness.

Viruses such as HIV, influenza, picornavirus and others are known stimulators of apoptosis. This individual cellular elimination is a preferential host defense in regenerative tissues. In contrast, if this death occurred in nonregenerating cells, such as neurons of the central nervous system, may result in disease. The target cell for rabies virus is the neuron. Here we studied the outcome of the interaction between rabies virus (CVS-11) and mouse brain cells. Replication of rabies virus in suckling mouse brain cells resulted in brain cell apoptosis, detected by DNA fragmentation and in situ apoptosis within 25 h after infection and before evidence of intracerebral immune activation. Cell death occurred simultaneously with rabies virus replication. There were clinical signs of illness in infected newborn mice within 24 h after the appearance of DNA fragmentation and before infiltration by lymphocytes. This suggested that onset of illness started independently of the immune function. This conclusion was supported by the occurrence of massive apoptosis followed by paralysis in rabies virus-infected immunosuppressed mice. Direct, viral-induced, neuronal apoptosis was the earliest death mechanism detected in these mice. We propose that pathogenesis of this fixed strain of rabies virus in mice begins with the induction of apoptosis by rabies virus replication. Cerebral damage may then be amplified by immunological mechanisms plus an additional unidentified factor. This is followed by increased permeability of the blood brain barrier.

The role of antibody in recovery from alphavirus encephalitis.

Alphaviruses infect neurons in the brain and spinal cord and cause acute encephalomyelitis in a variety of mammals. The outcome of infection is determined by whether the neurons survive infection and this, in turn, is determined by the virulence of the virus and the age of the host at the time of infection. We have been studying Sindbis virus (SV) infection of mice as a model system for alphavirus-induced encephalomyelitis. Investigation of intracerebral infection of weanling mice with two different strains of SV has allowed us to analyze the role of the immune response in protection from fatal disease (virulent NSV strain) and in clearance of virus from the nervous system during non-fatal disease (less virulent SV AR339 strain). Neutralizing and non-neutralizing antibodies to the E1 and E2 surface glycoproteins can protect mice from fatal NSV infection when given before or after infection, while T cells are not protective. The mechanism of antibody-mediated protection is not known, but it is likely that more than one mechanism is involved and that different mechanisms are involved in pre-infection and post-infection treatment protection. Clearance of infectious virus from the nervous system of mice during recovery from non-fatal disease is accomplished by antibodies to the E2 glycoprotein. The process does not involve damage to the infected neurons and is independent of complement and mononuclear cells. Bivalent antibody is required and binds to the surface of the infected cell. Initially, release of virus by budding from the cell surface is prevented and, subsequently, intracellular virus replication is inhibited possibly through antiviral mechanisms induced in co-operation with interferon. This non-lytic mechanism for control of virus infection results in the prolonged presence of viral RNA in tissue and the need for prolonged intrathecal synthesis of antiviral antibody by B cells within the central nervous system.

Temporal changes in chromatin, intracellular calcium, and poly(ADP-ribose) polymerase during Sindbis virus-induced apoptosis of neuroblastoma cells.

Sindbis virus (SV) induces apoptosis in many vertebrate cells, but the mechanism is unknown. To gain insight into this mechanism, the nature and time course of intracellular changes related to programmed cell death were studied in SV-infected mouse neuroblastoma cells. New virus production began at 5 h after infection and reach a peak at 12 h. Hoechst 33342 staining of DNA analyzed by flow cytometry demonstrated changes in chromatin beginning 6 h after infection. These chromatin changes were cell cycle dependent, affecting cells in G0/G1 but not S phase. Apoptosis was not dependent on increases in intracellular Ca2+ and occurred more rapidly in the absence of extracellular Ca2+. Nuclear changes were accompanied by activation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), resulting in increased consumption of NAD which was apparent by 10 h after infection. SV-induced apoptosis also involved the proteolytic cleavage of PARP. This cleavage was detectable at 16 h after infection approximately the same time that DNA fragmentation was apparent by agarose gel electrophoresis. We conclude that SV-induced apoptosis of neuroblastoma cells is dependent on viral replication, is not dependent on a rise in intracellular Ca2+, and is accompanied by activation of PARP and of a protease that cleaves PARP.

Roles of immunoglobulin valency and the heavy-chain constant domain in antibody-mediated downregulation of Sindbis virus replication in persistently infected neurons.

Clearance of infectious Sindbis virus from neurons is mediated by antibody to the E2 glycoprotein. Properties of the antibody important for downregulation of Sindbis virus replication are unknown. Immunoglobulin isotypes and valency determine many biological properties of antibodies. An immunoglobulin G1 (IgG1) isotype switch mutant and F(ab')2 and Fab fragments of IgG3 monoclonal antibody 209 were prepared and tested for clearance of infectious virus from persistently infected rat dorsal root ganglion neurons in vitro. IgG1, IgG3, and IgG3-derived F(ab')2 fragments were similarly efficacious, while IgG3-derived Fab fragments had no effect on virus replication. Cross-linking of Fab with secondary antibodies restored antiviral activity. Therefore, we found no evidence that IgG subclass plays a role in control of intracellular Sindbis virus replication. However, bivalency appears to be crucial for the ability of E2-specific IgG molecules to mediate clearance of infectious virus from neuron cells, suggesting that cross-linking of E2 molecules is essential.

Neurovirulent strains of Alphavirus induce apoptosis in bcl-2-expressing cells: role of a single amino acid change in the E2 glycoprotein.

The isolation and sequence comparison of avirulent and neurovirulent strains of polio virus, alpha virus, herpes virus, immunodeficiency virus, and other viruses have identified genetic changes that are required to cause disease in the nervous system. The molecular mechanisms by which these genetic changes result in neurovirulence are unknown. An avirulent laboratory strain of the Alphavirus Sindbis kills most cultured cell lines not by lethal parasitism, but by inducing apoptosis or programmed cell death. Transfection of cultured cells with the human bcl-2 oncogene can block Sindbis virus-induced apoptosis, resulting in a persistent viral infection resembling that observed in brains of immunodeficient mice. We investigated the possibility that neurovirulent strains of Sindbis virus could overcome the protective effects of bcl-2--a potential mechanism to explain the ability of these strains to cause fatal disease. Strains of Sindbis virus that were lethal for 2- to 4-week-old mice induced apoptotic death in cultured cells despite the presence of bcl-2. Using recombinant viruses, we show that a single amino acid change in the E2 glycoprotein of Sindbis virus confers both neurovirulence and the ability to kill cells expressing bcl-2.

The effects of alphavirus infection on neurons.

Sindbis virus is an alphavirus that causes encephalitis in mice. The primary target cells for central nervous system infection are neurons. The outcome of neuronal infection is dependent on the age of the mouse at the time of infection (maturity of the neuron) and the strain of virus used for infection (virulence of the virus). Sindbis virus causes neuronal death by inducing apoptosis. As neurons mature, they become resistant to virus-induced apoptosis, resulting in a persistent infection. Host production of antibody to a viral surface glycoprotein acts to downregulate virus replication in the infected neurons by a noncytolytic mechanism and clears infectious virus from the central nervous system. Specific genetic changes in the virus result in more virulent strains that cause severe disease and sometimes death in mature animals. These same genetic alterations also confer the ability to overcome the resistance of neurons to induction of cell death. Therefore, mature neurons infected with virulent viruses do not recover from infection even in the presence of an adequate immune response.

Adenovirus early region 4 and viral DNA synthesis.

Mutants of human adenovirus type 5 (Ad5) lacking early region 4 (E4) display a complex phenotype that includes a delay in the onset of viral DNA replication in low-multiplicity infections. Studies of viral DNA replication in vitro have not revealed a requirement for E4 products in DNA synthesis and, for most E4 mutants, defects in DNA replication are not apparent at high multiplicities of infection. The effects of E4 mutations on DNA replication therefore may reflect a role for E4 in the regulation of replication rather than in the process of DNA synthesis. The E4 mutant H5d/1014 carries two deletion mutations that together destroy all E4 open reading frames (ORFs) except ORF 4. Immunoprecipitation measurements of the level of the ORF 4 product confirm that H5d/1014 accumulates the ORF 4 product in somewhat larger amounts than wild-type Ad5. H5d/1014 is profoundly defective in viral DNA replication at a multiplicity of infection (50 PFU/cell) and time (24 hr after infection) that permit mutants lacking all seven E4 products to accumulate approximately normal amounts of DNA. In contrast, H5d/1019, a derivative of H5d/1014 in which the expression of ORF 4 is prevented by a mutation in the ORF 4 ATG initiator codon, produces DNA normally under these conditions. The product of ORF 4 therefore is necessary for the inhibition of viral DNA replication in H5d/1014-infected cells. H5d/1014 also inhibits, in trans, the synthesis of viral DNA by other E4 mutants that lack both E4 ORFs 3 and 6. Viruses that possess either of those ORFs are not subject to inhibition, indicating that the ORF3 and 6 products antagonize the effect of ORF 4. These observations are consistent with a regulatory role for the E4 ORF 3, 4, and 6 products in viral DNA replication in adenovirus-infected cells.

Identification of a putative alphavirus receptor on mouse neural cells.

Alphaviruses replicate in a wide variety of cells in vitro. The prototype alphavirus, Sindbis virus, causes an age-dependent encephalitis in mice and serves as an important model system for the study of alphavirus neurovirulence. To begin to understand the role of cellular virus receptors in the pathogenesis of Sindbis virus infection, we developed an anti-idiotypic antibody made in rabbits against a neutralizing monoclonal antibody specific for the E2 surface glycoprotein. The anti-idiotypic antibody (anti-Id 209) bound to N18 mouse neuroblastoma cells and inhibited adsorption of 35S-labeled virus by 50%. Binding of anti-Id 209 was inhibited by pretreatment of N18 cells with various proteases but not with neuraminidase or phospholipase, while virus binding was inhibited by pretreatment with phospholipase as well as protease. Anti-Id 209 precipitated proteins of 110 and 74 kDa from N18 cells intrinsically labeled with [35S]methionine. N18 cells grow with two phenotypes in culture, and immunoprecipitation of 125I-surface-labeled cells showed that the 74-kDa protein was present on loosely adherent cells growing in aggregates, while the 110-kDa protein was present in smaller amounts on firmly adherent cells growing as a monolayer. Analysis of brain cells from newborn mice by flow cytometry showed that all cells expressed the receptor protein at birth, but by 4 days after birth half of the cells had ceased receptor expression. A survey of other cell lines showed the protein to be present on murine fibroblastic and other rodent neuroblastoma cell lines but rarely on human neural or nonneural cell lines. These studies suggest that one of the receptors for Sindbis virus on mouse neural cells is a protein that is regulated during development of the nervous system. Developmental down-regulation of receptor protein expression may contribute to the age-dependent nature of susceptibility of mice to fatal alphavirus encephalitis.

Antibody to peptides of human myelin basic protein in post-rabies vaccine encephalomyelitis sera.

Development of neurologic complications after Semple rabies vaccine is closely linked to development of antibody to myelin basic protein (MBP). The portions of MBP against which the antibodies are directed were analyzed by enzyme immunoassay in sera and cerebrospinal fluid from 27 patients with vaccine complications. Most of the antibody was directed to regions of MBP peptides 45-89 and 90-170. There was no apparent correlation between antibody specificity for MBP peptides 1-44, 45-89 and 90-170 and the type of post-vaccinal neurologic complication. We conclude that the immunoglobulin repertoire in human B lymphocytes for responding to human MBP favors the portion of the MBP molecule containing residues 45-170.

Humoral and cell-mediated immune responses to various economical regimens of purified Vero cell rabies vaccine.

Purified Vero cell rabies vaccine (PVRV) is a new effective but inexpensive tissue culture rabies vaccine for human use. We investigated if the cost of immunization with PVRV could be further reduced by intradermal immunization. Fifty-eight subjects with low-risk exposure to rabies were randomized into 4 groups to receive full-dose (0.5 ml) intramuscular injection of PVRV on days 0, 3, 7, 14 and 28 or 4, 2 or 1 intradermal injections of PVRV (0.1 ml) on days 0, 3, and 7, followed by another intradermal injection on day 28. Neutralizing antibodies and specific cell-mediated response (CMIR) were sequentially followed up to day 36. The antibody levels in the intradermal groups increased with the number of injection sites and the levels achieved by the 2-site i.d. regimen were not significantly different from those obtained by the full-dose i.m. even though only 1/3 of the amount of PVRV was used. Specific CMIR occurred 1 week sooner in the 2 and 4-site i.d. regimens than the full-dose i.m. We therefore recommended that our 2-site i.d. regimen of PVRV should be further tested with a view to substituting it for the more expensive full-dose i.m. regimen in order to further reduce the cost of rabies prophylaxis particularly in the developing countries.

Anti-HIV positivity in Thailand: the usefulness of another ELISA test kit and Western blot as confirmatory tests.

Sera from 47 individuals repeatedly reactive in one screening ELISA system (designated as ELISA-A) for antibodies against human immunodeficiency virus (HIV) were evaluated by a second ELISA system (designated as ELISA-B) as well as by the Western blot technique. Both ELISA systems and the Western blot were positive in all of the 14 patients with clinical diagnoses of AIDS and AIDS-related persistent generalized lymphadenopathy (PGL). Of the 7 asymptomatic gays whose sera were repeatedly reactive in ELISA-A, 5 were also reactive in ELISA-B and these were the ones with positive Western blot tests. Eight and 17 ELISA-A reactive individuals were uncovered during a survey of 2,699 female prostitutes and 15,210 potential workers for Saudi Arabia respectively. All of these 25 individuals were ELISA-B and Western blot negative, an indication of false-positive reactivity with ELISA-A. Our studies indicate that the prevalence of HIV infection among the general Thai population is still low, and that the specificity of two ELISA test kits for anti-HIV may differ considerably. We concluded that evaluation of test kits should include studies in tropical countries where ecological conditions, climate and background endemic disease patterns are different than in the countries producing the diagnostic systems. Such studies are needed to identify the most sensitive and specific kits for worldwide application. We did discover that concordant positivity of two different ELISA test kits served as a reliable and inexpensive confirmatory test for anti-HIV.

An effective economical intradermal regimen of human diploid cell rabies vaccination for post-exposure treatment.

A closely-spaced multisite intradermal regimen of human diploid cell rabies vaccine (HDCV) was evaluated in 39 patients after low-risk exposure to rabies, in comparison to full-dose intramuscular HDCV and sheep brain-derived rabies (Semple) vaccine. The regimen consisted of four intradermal injections, 0.1 ml each of HDCV on days 0, 3 and 7, followed by two booster doses of only 0.1 ml each on days 28 and 91 administered intradermally. Although the total amount of HDCV used in this intradermal regimen was 1.4 ml or one-quarter of the conventional intramuscular regimen, a higher proportion of the recipients of this economical intradermal regimen, as compared to the full-dose intramuscular regimen, developed neutralizing antibodies above the hypothetical protective level of 0.5 iu/ml 7 days after starting immunization. Besides the earlier antibody response, the peak antibody level of the intradermal regimen was also satisfactorily high and not significantly different from that after the intramuscular regimen. Simultaneous administration of inosiplex, an antiviral and immunopotentiating agent, during the first 10 days of intradermal immunization resulted in an even higher antibody response for as long as 91 days. In contrast, but not unexpectedly, Semple vaccine evoked lower, more sluggish and inconsistent antibody responses. The side-effects of intradermal HDCV were mild, mainly local and self-remitting. We therefore recommend our intensive intradermal regimen of HDCV vaccination for safe, effective and economical use in post-exposure rabies immunization.

Cell-mediated immune response following intracutaneous immunisation with human diploid cell rabies vaccine.

Specific cell-mediated immune response (CMIR) against rabies antigens was studied in recipients of two regimens of human diploid cell rabies vaccine (HDCV) using the antigen-stimulated lymphocyte transformation test (LTT) as a measure of CMIR. Reconstituted HDCV could be conveniently used as the in vitro stimulating antigen and the response was antigen-dependent. Conventional intramuscular immunization with full-dose HDCV resulted in positive LTT as early as 14 days after starting immunisation, and peaked on day 28. Intracutaneous immunisation with 0.1 ml of HDCV at four sites on days 0, 3 and 7 was a more efficient means of inducing specific lymphocyte response. Specific CMIR was evident as early as seven days and became maximal on day 14. In addition to the more rapid induction of specific CMIR, our intracutaneous regimen also resulted in a brisker and higher antibody response than the intramuscular regimen. The peak antibody level of the intracutaneous regimen was reached on day 14 whereas that of the intramuscular regimen was reached on day 28 and the geometric mean antibody titre on day 14 of the intracutaneous route was significantly higher than that of the intramuscular regimen. We therefore conclude that our closely spaced intracutaneous immunisation with HDCV was effective both in the induction of specific antibodies and the cell-mediated immune response.