Immunization with a human diploid cell strain of rabies virus vaccine: two-year results

Antibody responses following primary vaccination with 1.0 ml of intramuscularly (im) or 0.1 ml of intradermally (id) administered human diploid cell rabies virus vaccine were observered for two years. Three primary doses of vaccine were given to 77 volunteers on days 0, 28, and 56. An antibody response was detected in all vacinees after a single dose; at one month, the response in the group that received vaccine id was identical to that in the group that was given vaccine im, although only 1/10th of the dose of vaccine was used. After the second and third doses, the antibody responses were higher with the primary im regimen; this difference was significant at two, three, and 12 months when the geometric mean titers of antibody were twofold higher for im than for id vaccination. The antibody responses to a booster dose of vaccine administered to randomly grouped volunteers by the subcutaneous or id route at six, 12, or 24 months were similar irrespective of the method of primary immunization but were greater with increasing intervals between primary and booster doses.     

Antibody responses to human diploid cell vaccine for rabies with and without human rabies immune globulin

One hundred one volunteers with no exposure to rabies were given human diploid cell vaccine (HDCV) for rabies with or without 20 international units of human rabies immune globulin (HRIG)/kg of body weight to evaluate schedules for therapy with HDCV and HRIG after exposure. All of the volunteers who received three or more doses of HDCV alone or four or more doses of HDCV with HRIG developed high titers of neutralizing antibodies by day 35, which persisted for at least 60 days. By day 7, of the 61 volunteers given HRIG and HDCV, 53% had neutralizing antibodies by a mouse neutralization test and 67% had neutralizing antibodies by a rapid fluorescent focus inhibition test. Similar antibody levels were found in volunteers given HRIG alone, a finding which suggests that low or undetectable early titers after administration of HDCV and HRIG were due to inadequate HRIG dosage rather than any interaction between the passive antibody (HRIG) and the vaccine antigen. These results suggest that trials with 30 or 40 international units of HRIG/kg in combination with HDCV are warranted.     

Pre-exposure rabies immunization with human diploid cell vaccine: decreased antibody responses in persons immunized in developing countries

In November 1982, a U.S. Peace Corps volunteer in Kenya completed pre-exposure rabies prophylaxis with a standard 3 dose intradermal (ID) series of human diploid cell rabies vaccine (HDCV). In May 1983, she was bitten by a dog and died of rabies 3 months later. An initial investigation revealed that the patient, as well as 9 of 11 others immunized at the same time, had no rabies antibody titers (less than 1:5). We therefore instituted investigations into the immunogenicity of pre-exposure HDCV both in the United States and in developing countries. A serosurvey revealed unexpectedly low rabies titers in both Peace Corps volunteers and others immunized in developing countries. Antibody titers measured 2-3 weeks after ID immunization were compared in 9 groups totaling 271 persons in the United States and Kenya. There was no statistically significant difference in antibody titers in the 6 U.S. groups immunized from 1980-1984 (P greater than 0.15); however, groups immunized in the United States had significantly higher titers than a group of Kenyan nationals (P less than or equal to 0.0001), and the Kenyans had significantly higher titers than 2 Peace Corps groups immunized in Kenya (P less than or equal to 0.0001). No single hypothesis proposed (laboratory error, vaccine potency, vaccination technique, or specific immune suppression) accounted for the observed differences. Although we cannot fully explain the poor response to HDCV, it is probably due to multiple factors. We conclude that persons immunized with ID pre-exposure HDCV in developing countries should have rabies antibody titers determined to ensure their seroconversion; for persons immunized in the United States, such titers need not be routinely determined.     

A new Vero cell rabies vaccine: results of a comparative trial with human diploid cell rabies vaccine in children.

We evaluated the immunogenicity and safety of a chromatographically purified rabies vaccine (CPRV) compared with human diploid cell rabies vaccine (HDCV) after pre-exposure immunizations (both primary and booster). Intramuscular doses of either 0.5 mL of CPRV or 1.0 mL of HDCV were given to 400 schoolchildren on days 0, 7, 28, and 365 (booster). Adequate titers of antibody (> or = 0.15 IU/mL, as defined by the Centers for Disease Control and Prevention) were observed in serum samples from all children 14 days after primary immunization with CPRV and HDCV; the antibodies persisted in all but one child up until 1 year. Fourteen days after the primary immunization series (day 42) and 7 days after booster immunization (day 372), all children had antibody titers of > or = 0.5 IU/mL. Local and systemic reactions after primary and booster immunizations occurred significantly less frequently in the CPRV group. A severe allergic reaction (angioedema) was reported in only one child after booster immunization with HDCV. CPRV has adequate immunogenicity for primary and booster pre-exposure immunizations in children and has a better safety profile than does HDCV.     

Effect of inosiplex on the humoral and cell-mediated immune responses to intradermal human diploid cell rabies vaccine

Antigen-stimulated lymphocyte transformation was studied in recipients of intradermal human diploid cell rabies vaccine (HDCV). HDCV was administered intradermally at 8 different anatomical sites, 0.1 ml each, on day 0; followed by another 4-site injection on day 7. Rabies antigen-stimulated in vitro proliferative response was evident as early as 7 days after starting immunization. It reached a peak on day 14 and had declined by day 28. The cellular proliferative response preceded and roughly correlated with the antirabies antibody response. Simultaneous administration of inosiplex, an antiviral and immunopotentiating drug, during the first 10 days of intradermal HDCV immunization did not result in heightened antibody titres or cell-mediated immune response to the vaccine. The number of T cells and the lymphocyte proliferative response to phytohaemagglutinin in inosiplex-treated vaccinees were similarly not significantly different from untreated controls. Our results confirm other previous findings that a specific cell-mediated immune response can be consistently and rapidly induced by an intradermal regimen of HDCV immunization. The addition of inosiplex to this regimen did not enhance the humoral or cell-mediated immune responses to the vaccine. The apparent lack of immunostimulating effect of inosiplex in this setting may be the result of several factors such as the immunization schedule and the immunologic parameters examined.     

Intradermal immunization with reduced doses of human diploid cell strain rabies vaccine: evaluation of antibody response by ELISA and mixed hemadsorption test

The antibody response to the rabies human diploid cell strain vaccine was compared after pre-exposure immunization with 0.1 ml intradermal (i.d.) and 1.0 ml subcutaneous (s.c.) vaccination. After primary immunization, 2 doses of vaccine 1 month apart, all vaccinees in the two groups had antibody titers detectable by ELISA. However, the mean antibody titer after the 0.1 ml i.d. doses (5.9 EU/ml) was half of what was obtained after the 1.0 ml s.c. doses (12.2 EU/ml). Likewise, though all vaccinees responded on the 1-year booster dose the mean antibody level after i.d. vaccination (8 EU/ml) was 2.5 times lower than after 1.0 ml s.c. dose (21.5 EU/ml). At 1 year 70% of the vaccinees had detectable antibody levels irrespective of vaccination route. A few individuals responded to one of their i.d. doses with only minor titer rises which is supposed to be due to inadvertent s.c. injection of the i.d. dose.     

Rabies preexposure prophylaxis with human diploid cell rabies vaccine: a dose-response study

Recent studies suggest that human diploid cell rabies vaccine (HDCV) may not always produce acceptable titers after intradermal (id) preexposure prophylaxis. To stimulate accidental deviation from the recommended route of administration and to determine the immunogenicity of smaller-than-recommended doses of HDCV, we injected each of 154 persons either intramuscularly (im) with 100%, 10%, or 3% of the standard im dose of vaccine or id with 10%, 3% or 1% of the standard im dose. Seroconversion (titers of antibody greater than or equal to 1:11) was found in all subjects at 49 and 90 days after vaccination. Titers were higher for subjects receiving 100% of the recommended dose im than for those receiving 10% of this dose id (P less than .01); these titers in turn were higher than those from persons receiving smaller doses (P less than .05). Persons receiving 10% or 3% of the standard im dose had lower titers on day 49 than did those receiving the same dose id (P less than .05).     

Confirmed rabies exposure during pregnancy: treatment with human rabies immune globulin and human diploid cell vaccine

A review of the literature shows 24 cases of pregnant human exposure to rabies virus through confirmed rabid animal bites. Historically, these patients received passive immunization with equine rabies immunoglobulin and/or purified vero cell vaccine or duck embryo vaccine. With the recent development of human-derived rabies vaccines, we report an additional case of human gestational rabies exposure, which was treated with human rabies immune globulin and human diploid cell vaccine.     

The early kinetics of the neutralizing antibody response after booster immunizations with human diploid cell rabies vaccine

Persons immunized in developing countries were recently shown to have low titers after pre-exposure immunization with human diploid cell rabies vaccine (HDCV). An investigation into the response to HDCV boosters was conducted to determine if immunologic sensitization had occurred and if there was a response difference in persons immunized in and outside of the United States. Intramuscular (im) booster doses of vaccine were administered to 113 persons previously immunized outside the United States and 47 persons immunized in the United States. The post-exposure booster regimen of a single 1.0-ml im booster, as recommended by the World Health Organization for all but the most severe bites, produced a one-dilution (5-fold) rise in antibody titer in 14 (11%) of 123 persons tested 5 days after booster and in 56 (89%) of 63 persons studied 7 days after booster. Persons immunized in the United States and those immunized outside the United States had similar responses. Persons with low pre-booster titers were more likely to exhibit a 5-fold rise in antibody titer 5 days after booster (P = 0.03) than persons with higher pre-booster titers. The post-exposure booster regimen of 2 1.0-ml im doses (one each on days 0 and 3), recommended in the United States, produced a more rapid response than the single booster regimen in only some persons; a 5-fold response occurred in 6 (50%) of 12 persons 5 days after booster.(ABSTRACT TRUNCATED AT 250 WORDS)     

The primary hamster kidney cell rabies vaccine: adaptation of viral strain, production of vaccine, and pre- and postexposure treatment

The hamster kidney cell rabies vaccine was investigated as a substitute for classical nervous tissue rabies vaccine. The Beijing strain of fixed rabies virus was adapted to primary hamster kidney cells (PHKCs), and four types of rabies vaccine (plain, adjuvant, concentrated, and concentrated adjuvant vaccines) were developed for human use. The potencies of the vaccines met the requirements of the World Health Organization, and these vaccines elicited rather satisfactory antibody responses in volunteers. The postexposure use of vaccine was evaluated in 301 individuals, 97 of whom had been bitten by proven rabid animals. None of the individuals contracted rabies during the observation period. After several years of field trials with both pre- and postexposure vaccines, the evidence indicates that the PHKC rabies vaccines are effective and safe for human use.     

Early lymphocyte activation in elderly humans: impaired T and T-dependent B cell responses.

Immunosenescence is characterized by an increase in autoantibody production. Because both T and B cell stimulation are key events for producing antibodies, we investigated early T and B cell activation by means of CD23 and CD40L (two very early activation antigens). PBMC from elderly humans (EH) were studied following culture with either medium, anti-CD3mAb, rIL-4, or PMA + ionomycin. CD23 expression on elderly B cells after anti-CD3 challenge of PBMC, a reflect of T-dependent B cell activation, was clearly defective. Conversely, CD23 expression on EH B cells following activation with soluble factors as rIL-4 was preserved. CD40L expression was also impaired in EH T cells following anti-CD3 challenge. However, activation by means of PMA and/or ionomycin was preserved both in T cells (CD40L expression) and in B cells (CD23 expression). These results indicate that a defective T-dependent B cell activation related to defective T cell activation located between surface membrane and PKC/ionomycin function is an intrinsic characteristic of immunosenescence. We have not found intrinsic B-cell defects, and we conclude that the characteristically impaired early B cell activation in EH is mostly due to T cell defects.     

Antigenic relationship between human coronavirus strain OC 43 and hemagglutinating encephalomyelitis virus strain 67N of swine: antibody responses in human and animal sera.

Hemagglutinating encephalomyelitis virus of swine (HEV) was adapted to growth in suckling mouse brain. Electron micrographs of HEV-infected suckling mouse brain, prepared by negative staining and thin-section techniques, exhibited typical morphological characteristics shared with other members of the Coronaviridae. The adaptation of HEV to suckling mouse brain facilitated serologic testing by the use of common host reagents and compatible animal systems. With hemagglutination inhibition, complement-fixation, and neutralization tests, an antigenic relationship was demonstrated between human coronavirus OC 43 and HEV in specific immune and hyperimmune animal sera. Children and adults with seroconversion to OC 43 antigen had diagnostic rises in titer of antibody to HEV antigens. Individuals with seroconversion to human coronaviruse 229E and B814 demonstrated antibody to HEV but not diagnostic rises in titer. Swine with titers of antibody to HEV had lower or no detectable titers of antibody to coronavirus OC 43. Although the prevalence and geometric mean titer of antibody to OC 43 were higher than the titer of antibody to HEV in every group of normal humans tested, significant differences in antibody response to coronavirus OC 43 and HEV were seen between populations that did or did not have possible contact with swine. The evidence suggested that antibody to HEV in humans probably represented a heterologous response to infection with coronavirus OC 43. However, a heterotypic response to unknown or uncharacterized strains of coronavirus cannot be excluded.     

A new human male diploid cell strain, TIG-7: its age-related changes and comparison with a matched female TIG-1 cell strain

A new human diploid cell strain, TIG-7, which has the male karyotype, was established and characterized. Isozyme and histocompatibility typing of the cell strain was performed. The average in vitro life span of the cells is 73 population doublings. Changes in cell volume, doubling time, saturation density, the efficiency of cell attachment, plating efficiency, and relative DNA content were examined during in vitro cellular aging. Hydrocortisone slightly prolongs the life span of the cell strain when the hormone is administered to the cultures during middle passages. The age-related changes in the parameters of TIG-7 are not appreciably different from those of the previously established TIG-1 cell strain. These results show that this cell strain is useful for research on cellular aging; further profit is anticipated from research using a combination of these two sexually different cell strains.     

Prevalence, clinical significance, and strain specificity of neutralizing antibody to the human immunodeficiency virus

A semiautomated microtiter assay has been developed to quantitate neutralizing antibody to the human immunodeficiency virus. This assay has been found to be highly specific. Forty-four sera that were negative by enzyme-linked immunosorbent assay (ELISA) were tested under code: 42 were negative (less than 1:2), and 2 had titers of 1:2. By contrast, of 178 sera positive by western blot, 92.7% had detectable neutralizing antibody, and 12.5% had titers greater than or equal to 1:128. Neutralizing antibody titers correlated poorly with clinical diagnosis and T4/T8 ratios. Different isolates differed quantitatively in their sensitivity to neutralization by antibodies obtained from different patients; however, all strains tested so far have been neutralizable by all the sera tested. Neutralizing antibody titers correlated weakly, if at all, with direct or competition ELISA titers.     

Human lymphocyte antigens: production of a monoclonal antibody that defines functional thymus-derived lymphocyte subsets.

A monoclonal mouse antibody (3A1) that specifically bound to 65% of human peripheral blood (PB) thymus-derived (T) cells but did not bind to complement receptor-positive PB bone marrow-derived (B) cells, polymorphonuclear leukocytes, or human erythrocytes has been produced. The 3AI antibody was synthesized by a stable cloned lymphocyte hybrid cell line. This lymphocyte hybrid line (3AI) was derived from fusion of P3 X 63/Ag8 myeloma cells and spleen cells from BALB/c mice immunized with HSB-2 cells, a human T cell line. The 3A1 lymphocyte hybrid line produced mouse ascites fluid containing 3A1 antibody in saturating titers of up to 1:25,600. Purified PB T cells that carried the 3A1 antigen incorporated tritiated thymidine maximally in response to phytohemagglutinin and concanavalin A stimulation, whereas purified PB T cells that lacked the 3A1 antigen responded suboptimally to phytohemagglutinin and minimally to concanavalin A. Thus, the 3A1 antibody can be easily used to study the role of 3A1-positive and negative T cell subsets in the regulation of normal and abnormal human immune responses.     

Modulation of synovial cell products by a factor from a human cell line: T lymphocyte induction of a mononuclear cell factor.

A human cell line (U937) can be stimulated to produce a soluble factor (MCF) by either lectin-activated T lymphocytes or their soluble products. In prior studies, we showed that MCF produced by peripheral blood mononuclear cells can increase production of collagenase and prostaglandin E2 by adherent synovial cells obtained from enzymatically dispersed rheumatoid synovium. We show here that peripheral blood T lymphocytes or cloned human T lymphocyte lines are capable of inducing MCF production by the monocyte-like U937 cells. MCF can be demonstrated in the supernatant fluid from cocultures of U937 cells and T lymphocytes that have been stimulated with phytohemagglutinin or concanavalin A for 24-48 hr. In addition, the supernatant fluid from 24-hr lectin-stimulated T lymphocytes can be transferred onto the U937 cells and subsequently, MCF activity can be recovered from the U937 culture medium. The activity of the soluble T cell product on the U937 cells is both time- and dose-dependent. A human cell line capable of MCF production in continuous culture has not been previously available. The use of a monocyte-like cell line (U937) and cloned T lymphocytes now makes it possible to demonstrate the role of discrete cell populations in the production of MCF and other mediators.     

In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity.

A method is described that allows for the improvement of antibody affinity. This method, termed complementary-determining region (CDR) walking, does not require structural information on either antibody or antigen. Complementary-determining regions are targeted for random mutagenesis followed by selection for fitness, in this case increased binding affinity, by the phage-display approach. The current study targets a human CD4-binding-site anti-gp120 antibody that is potently and broadly neutralizing. Evolution of affinity of this antibody demonstrates in this case that affinity can be increased while reactivity to variants of human immunodeficiency virus type 1 is broadened. The neutralizing ability of this antibody is improved, as assayed with laboratory and primary clinical isolates of human immunodeficiency virus type 1. The ability to produce human antibodies of exceptional affinity and broad neutralizing ability has implications for the therapeutic and prophylactic application of antibodies for human immunodeficiency virus type 1 infection.     

Orientation of a human leukocyte interferon molecule on its cell surface receptor: carboxyl terminus remains accessible to a monoclonal antibody made against a synthetic interferon fragment.

An 125I-labeled monoclonal antibody made against a synthetic 56-residue fragment of human leukocyte interferon (IFN) alpha 1 recognizes human, Escherichia coli-derived IFN alpha A bound to the surface of Madin-Darby bovine kidney cells. A major fraction of the antibody recognizes IFN specifically bound to the cells, because the number of bound antibody molecules corresponds to the number of cell-bound IFN molecules (as measured with radiolabeled ligand) and because the fraction of the IFN unspecifically bound to the cells is less than 10% of the total bound IFN. A synthetic carboxyl-terminal 16-residue IFN peptide, though not inhibiting binding of IFN to cells, inhibits binding of antibody to IFN. A recombinant IFN alpha A molecule with a carboxyl-terminal 13-residue deletion, though still able to compete for binding of IFN to cells, is not recognized by the antibody. Scatchard plot analysis of the binding data revealed apparent dissociation constants of 6.0 x 10(-10) M for the antibody-IFN interaction and of 4.0 x 10(-11) M for the IFN-cell receptor interaction. The antibody inhibits the binding of IFN to cells only weakly and neutralizes the antiviral activity of the ligand only when in a large molar excess. We conclude that the carboxyl-terminal 10-16 residues that are predicted from the cloned IFN cDNAs and that are present in some natural IFNs are not involved in binding to cells but are antigenic and hence exposed on the molecules' surface. That the carboxyl terminus is not directly involved in binding to cells is consistent with the observation that some IFNs with carboxyl-terminal deletions are biologically active.     

Alteration of lymphocyte phenotype and function in sickle cell anemia: Implications for vaccine responses

Individuals with sickle cell anemia (SCA) have increased susceptibility to infections, secondary to impairment of immune function. Besides the described dysfunction in innate immunity, including impaired opsonization and phagocytosis of bacteria, evidence of dysfunction of T and B lymphocytes in SCA has also been reported. This includes reduction in the proportion of circulating CD4+ and CD8+ T cells, reduction of CD4+ helper: CD8+ suppressor T cell ratio, aberrant activation and dysfunction of regulatory T cells (T_reg), skewing of CD4+ T cells towards Th2 response and loss of IgM‐secreting CD27 + IgM^highIgD^low memory B cells. These changes occur on the background of immune activation characterized by predominance of memory CD4+ T cell phenotypes, increased Th17 signaling and elevated levels of C‐reactive protein and pro‐inflammatory cytokines IL‐6 and TNF‐α, which may affect the immunogenicity and protective efficacy of vaccines available to prevent infections in SCA. Thus, in order to optimize the use of vaccines in SCA, a thorough understanding of T and B lymphocyte functions and vaccine reactivity among individuals with SCA is needed. Studies should be encouraged of different SCA populations, including sub‐Saharan Africa where the burden of SCA is highest. This article summarizes our current understanding of lymphocyte biology in SCA, and highlights areas that warrant future research. Am. J. Hematol. 91:938–946, 2016. © 2016 Wiley Periodicals, Inc.     

Antigen-induced in vitro antibody production in humans: a model for B cell activation and immunoregulation

The precise events associated with B cell activation in humans are a subject of intense investigation. It has been difficult to develop an in vitro model of antigen-specific triggering of antibody synthesis by human peripheral blood mononuclear cells that is independent of exogenous mitogens. In the present study a sensitive and reproducible culture system and enzyme-linked immunosorbent assay have been established wherein antigen alone is used to trigger antigen-specific antibody synthesis by mononuclear cells from subjects immunized to keyhole limpet hemocyanin (KLH). The in vitro antigen-induced anti-KLH response is comparable in magnitude to that induced by pokeweed mitogen, is predominantly IgM in isotype, and is accompanied by a simultaneous increase in polyclonal antibody production. Anti-KLH responses were seen at in vitro KLH concentrations as low as 0.05 microgram/ml. However, concentrations of KLH greater than 5 microgram/ml resulted in profound suppression of the anti-LHL response while continuing to trigger large amounts of total polyclonal immunoglobulin synthesis. This suppression by high concentrations of antigen was also observed in pokeweed mitogen-driven anti-KLH production. These observations are consistent with previous results from the mouse model showing a close association between antigen-specific and polyclonal responses and the phenomenon of antigen-induced, antigen-specific suppression. Thus, an in vitro model of antigen induction of antigen-specific antibody synthesis in human peripheral blood mononuclear cells has been demontrated and should prove useful in exploring the mechanism of human B cell activation and immunoregulation.