ATTACHMENT OF CHRONIC LYMPHOCYTIC LEUKAEMIA CELLS BY AUTOLOGOUS POLYMORPHONUCLEAR NEUTROPHILS MEDIATED BY BISPECIFIC ANTI-CD19/CD64 ANTIBODY.

Chronic lymphocytic leukemia (CLL) is the most common adult leukaemia in the US and in Europe, including Georgia. CLL presents with clonal expansion and accumulation of CD5+CD19+CD23+ cells in peripheral lymphoid organs and tissues and in bone marrow. The disease remains incurable, albeit there are new molecular and immunotherapy methods currently available, which, in conjunction with chemotherapy, lead to the "precision therapy" approach. The majority of immunotherapies are based on the ability of therapeutic antibodies to mobilize anti-tumour potential of immune responses. Bispecific antibodies (BsAb) are also considered in the treatment of CLL, whereby phagocytic cells play a key effector role in the destruction of the target CLL cells. Anti-CD19/anti-CD64 BsAb binds to CD19 receptors on CLL cells and to CD64 receptors (FcγRI) on monocytes and activated polymorphonuclear neutrophils (PMNs), thus inducing phagocytosis of the leukaemic cells. The aim of this study was to evaluate the ability of anti-CD19/CD64 BsAb to enhance adherence of CLL cells by PMNs, intact or activated with G-CSF and IFNγ cytokines. Membranes of the isolated CLL cells of 16 patients were stained with Red Fluoresent Linker and CLL cells were co-incubated with isolated autologous PMNs, intact or pre-stimulated with G-CSF and/or IFNγ for 4h or 24h. The PMN/CLL cell adhesion was analyzed with the FACScan flow cytometer by gating on PMNs with adhered RFL-stained CLL cells. The results were heterogenous. Our data demonstrate that anti-CD19/anti-CD64 BsAb has limited capacity to enhance leukemic cell attachment by autologous PMNs. This could partially be explained by the remarkable intensity of spontaneous ability of PMNs to adhere to autologous CLL cells. Pre-treatment of PMNs from CLL patients with G-CSF and INFγ, alone or jointly did not enhance the adhesion of the leukaemic cells. Moreover, G-CSF and IFNγ joint effect led to the reduction in the adhesion capacity of the effector PMNs. It appears, that therapeutic effect of anti-CD19/anti-CD64 BsAb on enhancing attachment of leukaemic cells to PMNs in CLL patients is limited and its application should be based on the assessment of individual capacity of the patients' phagocytic cells.

ELEVATED LEVELS OF NATURALLY OCCURRING AUTOANTIBODIES TO HUMAN CHORIONIC GONADOTROPIN Β CORE FRAGMENT IN A FEMALE PATIENT WITH THYROID FOLLICULAR ADENOMA: CASE REPORT.

We report a case of a woman, who had an elevated levels of naturally-occurring autoantibodies to human chorionic gonadotropin (hCG) ß core fragment ( hCGßcf) one year prior to the development of thyroid follicular lesion. The patient underwent surgery and the histology report demonstrated that the lesion was a follicular adenoma. Further investigations of the role of naturally-occurring autoantibodies (NAAbs) to anti-hCGßcf in the pathogenesis of various tumours of thyroid gland might be useful in the development of novel diagnostic methods, using anti-hCGßcf NAAbs as a marker for the detection of unsuspected thyroid tumour.

NATURALLY-OCCURRING AUTOANTIBODIES TO HUMAN CHORIONIC GONADOTROPIN AND ITS SUBUNITS IN OVARIAN CYST PATIENTS.

Growing evidence supports the existence of immune-surveillance mechanisms in ovarian tumour patients, including autoantibodies to tumour associated and tumour specific antigens, tumour growth factors. Glycoprotein hormone human chorionic gonadotropin (hCG) and its hormone-specific hCGß subunit have been associated with epithelial tumours such as bladder, lung, oral/facial, breast, cervical, ovarian, vaginal, prostate, renal and pancreatic carcinomas. It is believed that hCG plays a role of an autocrine growth factor for tumor cells. Here we have demonstrated that sera of patients with ovarian cyst contain naturally-occurring autoantibodies, predominantly of IgG2 isotype, that bind to hCG and its subunits with high affinity. Titration of blood sera from 36 female patients, aged 22-61 after ethical permission and informed consent, diagnosed with ovarian cyst and healthy age-matched controls (n=12) was performed using a classical enzyme-linked immunosorbent assay (ELISA). Binding of the sera to the following antigens was tested: hCGαß, hCGß, hCGα, hCGß C-terminal peptide (hCGßCTP) and hCGß core fragment (hCGßCF). The same type of ELISA (with necessary modifications) was used for further investigation of subclass usage and assessment of binding affinity of the detected autoantibodies. Our data indicates that the sera of the majority of patients with ovarian cyst contain significantly higher levels of the natural IgG antibodies binding to hCGαß, hCGß, hCGα, hCGßCTP and hCGßCF, than those of the healthy controls. Natural IgG antibodies to hCGαß heterodimer were detected in 78% of cases, to hCGß in 61% of cases, to hCGα in 78% of cases, to hCGßCTP in 69% of cases, to hCGßCF in 83% of cases. These autoantibodies predominantly belonged to the IgG2 subclass and were characterized by the high binding affinity. It is plausible that they cross- bind to sugar side chains of hCG and its subunits. Our data demonstrated that sera of patients with the ovarian cyst contains elevated levels of naturally-occurring IgG antibodies, which bind to hCG and/or its subunits. The overwhelming majority of these autoantibodies belong to the IgG2 isotype thus indicating that they might be directed against carbohydrate antigens within highly glycosylated hCG.

Investigation of factors influencing the immunogenicity of hCG as a potential cancer vaccine.

Human chorionic gonadotrophin (hCG) and its ß-subunit (hCGß) are tumour autocrine growth factors whose presence in the serum of cancer patients has been linked to poorer prognosis. Previous studies have shown that vaccines which target these molecules and/or the 37 amino acid C-terminal hCGß peptide (hCGßCTP) induce antibody responses in a majority of human recipients. Here we explored whether the immunogenicity of vaccines containing an hCGß mutant (hCGßR68E, designed to eliminate cross-reactivity with luteinizing hormone) or hCGßCTP could be enhanced by coupling the immunogen to different carriers [keyhole limpet haemocyanin (KLH) or heat shock protein 70 (Hsp70)] using different cross-linkers [1-ethyl-3(3-dimethylaminopropyl)carboiimide (EDC) or glutaraldehyde (GAD)] and formulated with different adjuvants (RIBI or Montanide ISA720). While there was little to choose between KLH and Hsp70 as carriers, their influence on the effectiveness of a vaccine containing the BAChCGßR68E mutant was less marked, presumably because, being a foreign species, this mutant protein itself might provide T helper epitopes. The mutant provided a significantly better vaccine than the hCGßCTP peptide irrespective of the carrier used, how it was cross-linked to the carrier or which adjuvant was used when hCG was the target. Nonetheless, for use in humans where hCG is a tolerated self-protein, the need for a carrier is of fundamental importance. Highest antibody titres were obtained by linking the BAChCGßR68E to Hsp70 as a carrier by GAD and using RIBI as the adjuvant, which also resulted in antibodies with significantly higher affinity than those elicited by hCGßCTP peptide vaccine. This makes this mutant vaccine a promising candidate for therapeutic studies in hCGß-positive cancer patients.

PHAGOCYTOSIS AND EXPRESSION OF FCg-RECEPTORS AND CD180 ON MONOCYTES IN CHRONIC LYMPHOCYTIC LEUKEMIA.

Chronic lymphocytic leukaemia (CLL) is a lymphoproliferative disease characterised by accumulation of monoclonal CD19+CD5+CD23+ lymphocytes in the peripheral blood and bone marrow. CLL is the most common type of the adult leukemia in the Western world. The disease is incurable, albeit there are new molecular and immunotherapy methods currently available in conjunction with chemotherapy, leading to the "precision therapy". The majority of immunotherapeutic approaches are based on the ability of therapeutic antibodies (Rituximab, Alemtuzumab) to mobilize anti-tumour potential of the Natural Killer cells and macrophages/monocytes through their Fcg-receptors (FcγR). Therefore functional status of monocytes in CLL is an important contributor to the efficacy of this treatment. In addition, CLL patients are characterized by a profound immunodeficiency, and how this affects monocytes, has not been established. Here we study ex vivo phagocytic function and the expression of Fcg receptors and CD180 toll-like receptor (TLR) by monocytes of 14 untreated and 8 treated with Cyclophosmamide, Adriamycin, Prednisolone (COP) CLL patients, and 12 age-matched control volunteers. Phagocytic function was assessed through the ability of freshly isolated monocytes to attach and to engulf intact or opsonized Staphylococcus aureus particles in vitro with or without Granulocyte/Macrophage Colony Stimulating Factor (GM-CSF) and Interferon g (IFNg). Simultaneously, immunophenotyping for FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and CD180 has been carried out. The results were assessed by Flow Cytometry. Our results demonstrated that phagocytosis of the intact and opsonised or intact S. aureus by monocytes of CLL patients was significantly decreased in comparison with normal controls, with no recovery upon the treatment with GM-CSF and IFNg. A significant decrease in the expression of CD64 and CD180 has been detected on monocytes of CLL patients, with the drop in CD64 expression correlating with the disease progression and advanced Rai stages. In addition, the treatment with COP led to a more profound decrease in the expression of CD64. No appreciable changes were detected in the expression of CD32 and CD16 throughout the experiments. The diminished expression of CD64 and CD180 provides possible explanation for the impaired phagocytic function of monocytes in CLL, as FcγRI receptor interaction with opsonising IgG1, and CD180 with the ligands on S. aureus might affect the ability of monocytes to attach and to engulf S. aureus particles and effectively eliminate the pathogen. Our data indicates that the decreased functional status of monocytes in CLL might contribute to the diminished efficacy of therapeutic antibodies as well as to the immunodeficiency, characteristic for these patients.

Correlation of the expression of CD32 and CD180 receptors on CLL cells and MEC1 cell line.

Chronic Lymphocytic Leukemia (CLL) presents with clonal expansion and accumulation of CD5+CD19+CD23+ cells in peripheral lymphoid organs and tissues and in bone marrow. CLL is supposedly driven by exogenous and/or endogenous (auto)antigen(s) and there is increasing evidence that CLL cells receive microenvironmental signals which support their growth, survival and expansion in vivo. We have previously shown that powerful signals are received by CLL cells through CD180 orphan toll-like receptor. Additional accessory signals could be generated through FcγRII (CD32), since both are expressed on CLL cells as well as on control B cells. Here we studied correlation of the expression of CD32 and CD180 on CLL cells as well as on MEC1 cell line. Peripheral blood mononuclear cells (PBMC) from CLL patients and age-matched healthy volunteers were separated, stained with appropriate antibodies to CD19, CD32 and CD180 and analysed by flow cytometry. CD32 and CD180 expression on MEC1 cells was studied at different time-points. The data was statistically analysed using the Mann-Whitney non-parametrical test. Our data indicates that expression of CD32 is significantly increased on CLL cells compared to control B cells as well as in long-term MEC1 cell culture. In contrast, CD180 expression on MEC1 cells significantly decreased throughout 0-96h of MEC1 cell culture. We have recently shown that CD180 ligation can redirect sIgM-mediated signaling from pro-survival to pro-apoptotic. This data indicates that a drop in the expression of CD180 on cycling CLL cells might lead to a weakening of this effect and enhance further survival and expansion of CLL cells in proliferative centres of lymphoid tissues. Since MEC1 cells are derived from a CLL patient with mutated IGVH genes (M-CLL) negative correlation between CD180 and CD32 expression on cycling MEC1 cells could be limited to M-CLL.

Aberrant expression of Fcγ-receptors and Toll like receptor CD180 on monocytes from patients with chronic lymphocytic leukaemia.

Chronic lymphocytic leukemia (CLL) is the most common leukaemia in the US and in Europe, including Georgia. Patients with CLL are susceptible to infectious diseases as a result of both, the disease progression and chemotherapy that indicates deficiency of immune responses to pathogens, including innate immunity, mediated by monocytes. Monocytes are also often recruited by monoclonal antibodies (mAbs) which express anti-tumour toxicity through Fcγ-receptor (FcγR)-mediated phagocytosis of opsonised leukaemic cells. In this paper we address of monocytes functional status through assessment of the patterns of expression of Fcγ receptors CD64, CD32, CD16 and CD180 receptor on monocytes from CLL patients and healthy individuals using specific mAbs and flow cytometry. Our data demonstrate that monocytes from peripheral blood of CLL patients lack expression of CD64 and CD16 as well as CD180 that would substantially undermine their ability to contribute to anti-bacterial immune responses. In addition, aberrant expression of CD64 would negatively affect the efficiency of antibody-mediated immunotherapies.

Prevalence of high affinity naturally occurring IgG2 antibodies against human chorionic gonadotropin and its subunits in patients with ovarian cyst.

Naturally occurring antibodies to tumour antigens are gaining interest as clinically important cancer biomarkers for early diagnosis, prognosis and for the development of anti-cancer therapeutics. The glycoprotein αß heterodimer hormone human chorionic gonadotropin (hCG) and its ß subunit (hCGß) are produced by various cancers, and their increased serum levels correlate with poor prognosis. We have previously reported that patients with benign ovarian cysts, but not the malignant tumours, were characterized by augmented serum levels of naturally-occurring IgG antibodies to hCG and hCGß. Here we further characterise these antibodies in patients with ovarian cysts. IgG and IgM antibody binding to whole hCG, hCGß, hCGα, hCGß C-terminal peptide (hCGßCTP), and the hCGß core fragment (hCGßCF) were measured in the sera from 36 patients with ovarian cysts and 12 healthy non-pregnant women using a standard ELISA. IgG subclass usage and affinity was also determined together with cross-binding to whole hCG and its subunits of four selected commercial monoclonal antibodies generated against ovarian cyst mucins. Our results showed that 91.7% of the sera tested contained elevated IgG, but not IgM antibodies to one or several antigens, with an overwhelming prevalence of high affinity IgG2 indicating their binding to carbohydrate epitopes and possibly ovarian cyst mucins. Anti-mucin commercial antibody ab212418 (Abcam) produced against Gal1-3GalNAc, exhibited strong cross-binding to hCGαß, hCGß, hCGα and hCGßCTP. The protective anti-cancer potential of these antibodies will be further investigated and could lead to the development of novel treatment strategies for ovarian cancer.

CD5-induced apoptosis of B cells in some patients with chronic lymphocytic leukemia.

Although B chronic lymphocytic leukemia (B-CLL) is characterized by prolonged survival of CD5(+) B cells in vivo, these cells apoptose spontaneously in vitro. The effect of CD5 ligation on apoptosis was studied in 27 newly diagnosed patients with B-CLL, in relation to the expression of surface IgM (sIgM), CD79b, CD38, CD72 and CD19. B cells from 15 patients (group I) were resistant to anti-CD5-induced apoptosis, whereas apoptosis above spontaneous levels was seen in the remaining 12 studied (group II). Group II was then subdivided on the basis of differences in the time required to reach maximum apoptosis: whilst B cells from seven patients underwent apoptosis by 18 h, those from the remaining five needed 36 h to apoptose. The expression of sIgM, CD5, CD79b and CD38 was higher in group II than group I, suggesting that signaling for apoptosis might operate via CD79, and that CD38 expression was required. As shown by flow cytometry and confirmed by Western blotting, apoptosis was associated with a decrease in the ratios of Bcl-2/Bax and Bcl(XL)/Bax, due to an increase in the level of Bax, but no change in that of Bcl-2. This heterogeneous apoptotic response to CD5 ligation offers an explanation for the incomplete success of anti-CD5 monoclonal therapy, and might help identify patients who would respond to such treatment.

A low budget luminometer for sensitive chemiluminescent immunoassays.

We have designed a simple luminometer based on a reasonably priced Peltier-cooled charge-coupled device (CCD) camera, housed in a light-tight box, with straightforward lens imaging and a simple platform for a microtitre or other assay format. The quantitative readout of the CCD image is recorded on a PC using customised software. The instrument can be assembled in a standard university workshop for under pound3000, compared with the cheapest commercial instruments retailing at pound10,000 and above. Consistent with the general view on chemiluminescent assays, the sensitivity is 10-100 times greater than that obtained with parallel ELISA's using a chromogenic substrate. A unique feature of the CCD format is that it enables assays to be carried out on arrays of minidots and even nanodots of antigen on the floor of each microtitre well. This permits direct comparison and standardisation of reactivity of a single sample against several antigens and economy in the use of reagents, test sample and technician time; finger-prick samples of blood can be analysed. The instrument should have widespread applicability in developing countries and, indeed, in any laboratories with hard-pressed budgets.

Differential expression of CD 180 and IgM by B-cell chronic lymphocytic leukaemia cells using mutated and unmutated immunoglobulin VH genes.

We have studied the surface expression of the Toll-like receptor family member CD 180 on cells from 78 patients with B-chronic lymphocytic leukaemia (B-CLL). B-CLL cells had variable levels of CD 180 expression, but this was always less than that expressed by normal blood B cells and was stable for 24 months. Significantly higher levels of CD 180 were expressed by B-CLL cells with mutated IGVH genes compared with those using unmutated IGVH genes. This was in contrast to the higher levels of expression of surface immunoglobulin M by B-CLL cells using unmutated, rather than mutated IGVH genes. CD 180 was functional on B-CLL cells from some of the patients, as shown by the increased expression of CD 86 following incubation in vitro with anti-CD 180. The differential expression of CD 180 amongst B-CLL patients is one more marker that may define more precisely the different biological properties of this heterogeneous disease.

[Age-related changes in the immunoglobulins of human blood serum]. / Vozrastnye izmeneniia kontsentratsii immunoglobulinov v syvorotke krovi cheloveka.

It has been shown that the content of G and A immunoglobulin (IgG, IgA) in blood serum increases with human age. IgM quantity is maximum at child age and at old age (about 80 years old and elder), at the age of 15-20 it is minimum. Immunoglobulin concentration is higher in female's blood serum than in male's, particularly at middle and old ages. The role of X-chromosome in regulation of serum IgM concentration is being discussed.

Expansion of CD4+ T cells with a cytotoxic phenotype in patients with B-chronic lymphocytic leukaemia (B-CLL).

Abnormal CD4/CD8 ratios and T-cell function have previously been shown in patients with B-chronic lymphocytic leukaemia (B-CLL). We have demonstrated that CD4+ T cells containing both serine esterase and perforin (PF) are increased in the blood of these patients. Using flow cytometry, we have shown that the CD4+ PF+ cells were CD57+ but lacked expression of CD28, suggesting a mature population. The same phenotype in CD8+ T cells is characteristic of mature cytotoxic T cells. However, in contrast to the CD8+ T cells, the CD4+ T cells were more frequently CD45RO positive than CD45RA positive, indicating prior antigen experience. In contrast, this population lacked expression of either CD69 or HLA-DR, arguing that they were not activated or that they are an abnormal population of T cells. Their constitutive cytokine levels showed them mainly to contain IL4 and not IFNgamma, suggesting a Th2 phenotype. The role of the CD4+ PF+ T-cell population is at present uncertain. However, this potentially cytotoxic T-cell population could contribute both to enhancing survival of the B-CLL tumour cells through production of IL4, and to the immunodeficient state frequently seen in patients with this tumour, independent of drug treatment.

Recent progress in the understanding of B-cell functions in autoimmunity.

Our early concepts of the normal role of B cells in immunity focused on their ability to produce antibodies (Ab) and in the case of autoimmune diseases autoAbs, some of which were pathogenic. Over the past 10 years, it has became apparent that B cells display a variety of characteristics, other than Ab production, which could contribute to autoimmunity. They normally play a role in the development of lymphoid architecture, regulating T-cell subsets and dendritic cell (DC) function through cytokine production, and in activation of T cells. Receptors editing is also important in B cells which aids in immunity to infection and, possibly, prevention of autoimmunity. Transgenic animal models have now shown that B cells are necessary for many autoimmune diseases although their Ab products are not required in some cases. Negative signalling by CD5 and other molecules, such as CD22, in maintaining tolerance through recruitment of src-homology two domain-containing protein tyrosine phosphatase-1 has also been documented. In fact, we have now reached a new era whereby the B cell has returned as an important contributor to autoimmune disorders, so that the race is on to characterize signalling regulation via the B-cell receptor and coreceptors. Identification of such molecules and their potential defects should lead to effective ways of controlling the immune response and in particular preventing the development of autoimmune states. The classical view of B cells in the biology of immune responses to infectious and self-antigens (Ag) that they promote immunity primarily by producing Ab turns out to be rather naïve. Indeed, studies over the last few years indicate that this view is far from complete, and suggest that B lymphocytes have extraordinarily diverse functions within the immune system. Furthermore, it is becoming increasingly clear that the pathogenesis of autoimmune diseases cannot solely be accounted for by T cells, and intrinsic abnormalities of B cells have been described in such conditions. In this brief review we highlight some recent observations in the context of B lymphocyte in pathophysiology, and focus on their revival as pivotal players the pathophysiology in autoimmune diseases. Yet, it remains difficult to provide a model of how important B cells are in immunity and autoimmunity.

DNA vaccination favours memory rather than effector B cell responses.

Following priming and boosting of mice with a DNA vector pEE6DeltaS-hCGss expressing sequences encoding a transmembrane version of the beta-chain of human chorionic gonadotropin (hCGbeta), we failed to detect appreciable levels of specific antibody. However, subsequent challenge with hCG protein in Ribi adjuvant elicited a strong and rapid secondary immune response. This response was of comparable magnitude to that produced following priming, boosting and challenge with protein in adjuvant. Thus, DNA vaccination with this vector is as efficient in generating B cell memory as is conventional immunization, but the memory generation occurs in the absence of an overt effector response. Despite an overall similar level of specific antibody, the DNA-vaccinated mice produced hCG-specific antibodies biased towards IgG2a and IgG2b isotypes, whereas the protein-vaccinated mice produced higher levels of IgG1 antibodies. Both Th1 and Th2 cytokines (interferon-gamma (IFN-gamma) and IL-4) were lower in the spleens of the DNA-immunized animals compared with the protein-Ribi-immunized animals, possibly suggesting a different level of helper T cell response to the two different modes of immunization.

Epitopes of human chorionic gonadotropin and their relationship to immunogenicity and cross-reactivity of beta-chain mutants.

PROBLEM: Human chrionic gonadotropin (hCG) is a placental glycoprotein hormone, a heterodimeric molecule, consisting of alpha and beta chains. It induces the synthesis of progesterone, which is essential for the maintenance of the fertilized egg. Antibodies directed against hCG can, therefore, prevent pregnancy and serve as a vaccine. hCG belongs to the glycoprotein hormone family and shares the alpha chain with the other members. The beta chain is a hormone-specific subunit that is unique to hCG, but still possesses 85% amino acid homology with the beta chain of luteinizing hormone (LH), which means that prolonged immunization with hCG produces antibodies that cross-react with LH. METHOD OF STUDY: We have taken an approach involving the mutation of beta hCG to eliminate cross-reactive epitopes without affecting the natural folding of the polypeptide chain and thus the unique beta hCG-specific epitopes. RESULTS: Several mutants have been constructed that have maintained the binding to hCG-specific monoclonal antibodies (mAbs) but have lost the ability to bind to a panel of LH cross-reactive mAbs. To investigate the immunogenicity of selected mutants, mice were immunized with expression plasmid DNA, containing the gene for wild-type beta hCG and two mutants: mutant 3, with four amino acid substitutions (68 Arg-->Glu; 74 Arg-->Ser; 75 Gly-->His; 79 Val-->His), and mutant 7, with a single amino acid substitution (68 Arg-->Glu). CONCLUSIONS: Although both mutants were able to elicit antibody responses in at least some animals, the levels were less than those seen with the wild-type beta hCG DNA, and there seems still to be a residual cross-reactivity with LH. Attempts to improve the immunogenicity of the mutants and to further modify the sequence to remove the cross-reactivity are currently underway.

Refocusing of B-cell responses following a single amino acid substitution in an antigen.

Intranasal immunization of BALB/c strain mice was carried out using baculovirus-derived human chorionic gonadotrophin (hCG) beta-chain, together with Escherichia coli heat-labile enterotoxin. Gonadotrophin-reactive immunoglobulin A (IgA) was induced in a remote mucosal site, the lung, in addition to a systemic IgG response. The extensive sequence homology with luteinizing hormone (LH) results in the production of LH cross-reactive antibodies when holo-hCG is used as an immunogen. In contrast to wild-type hCGbeta, a mutated hCGbeta-chain containing an arginine to glutamic acid substitution at position 68 did not induce the production of antibodies which cross-react with LH. Furthermore, the epitopes utilized in the B-cell response to the mutated hCGbeta shifted away from the immunodominant region of the parent wild-type molecule towards epitopes within the normally weakly immunogenic C terminus. This shift in epitope usage was also seen following intramuscular immunization of rabbits. Thus, a single amino acid change, which does not disrupt the overall structure of the molecule, refocuses the immune response away from a disadvantageous cross-reactive epitope region and towards a normally weakly immunogenic but antigen-unique area. Similar mutational strategies for epitope-refocusing may be applicable to other vaccine candidate molecules.