Recombinant Major Surface Glycoprotein Research Articles

The role of LPD-nanoparticles containing recombinant major surface glycoprotein of Leishmania (rgp63) in protection against leishmaniasis in murine model

Context: Leishmaniasis is a major public health problem. Despite numerous attempts, yet there is no effective vaccine against human leishmaniasis, mainly due to a lack of an effective vaccine delivery system as well as adjuvant.Objective(s): The aim of this study was to evaluate the ability of recombinant glycoprotein 63 (rgp63) as a model of Leishmania antigen, entrapped in liposome–polycation–DNA (LPD) complexes nanoparticles in inducing cell mediated immune (CMI) response and protecting against L. major in BALB/c mice.Materials and methods: To this end, the abundant leishmania promastigote cell surface glycoprotein, gp63, was entrapped in nano-sized LPD (CpG) particles, (LPD (CpG)-rgp63), and BALB/c mice were immunized three times with either (LPD (CpG)-rgp63) or rgp63-CpG DNA or LPD (CpG) or free rgp63 and dextrose 5%. Various parameters including footpad thickness, splenic load of L. major parasites, rgp63-binding IgGs and also cytokine levels of rgp63-reactive T lymphocytes were then compared among different vaccinated animals.Results: The lowest number of parasites in spleen, the higher levels of IgG2a after challenge infection, the minimal footpad swelling and high level of IFN-γ secretion, all indicated that adjuvants and antigen-delivery systems are essential in modifying immune responses; as mice received LPD (CpG)-rgp63 induced immune response stronger than the other groups.Conclusions: This study demonstrates that LPD nanoparticle is a promising and adaptable delivery system which could be modified towards specific vaccine targets to induce a more potent immune response in combination with rgp63.

The role of liposome size on the type of immune response induced in BALB/c mice against leishmaniasis: rgp63 as a model antigen

To develop an efficient liposomal vaccine delivery system, the size of liposomes is critical to their adjuvant activities. In the present study, liposomes with different sizes (100, 400, 1000 nm) containing recombinant major surface glycoprotein of Leishmania (rgp63) were prepared, characterized, and inoculated subcutaneously into BALB/c mice to evaluate the rate of protection and the type of immune response generated against leishmaniasis. The lowest footpad lesion size and splenic parasite burden were seen in the mice immunized with large size (≥400 nm) liposomes after challenge with Leishmania major. The production of IFN-γ was only elevated in the spleen cells of mice immunized with large size (≥400 nm) liposomes. The highest IgG2a/IgG1 ratio was also seen in the sera of the mice immunized with large size (≥400 nm) liposomes before and 14 weeks after challenge. The results showed that immunization with small size (100 nm) liposomes induces a Th2 response, whereas immunization with large size (≥400 nm) liposomes induces a Th1 type of immune response. There was no significant difference in the type of induced immune response between the mice immunized with liposomes of 400 nm and those immunized with liposomes of 1000 nm or unextruded. The results of the current study demonstrated that the size of liposomes plays a significant role in the type of generated immune response.

Evidence for high prevalence of Pneumocystis jirovecii exposure among Cameroonians

Cameroon lacks the capacity for routine Pneumocystis pneumonia (PcP) diagnosis, thus, the prevalence of Cameroonian exposure to this microbe is unknown. It is known that Pneumocystis infecting different mammalian host species represent diverse phylogenetic backgrounds and are now designated as separate species. The highly sensitive nature of ELISA and the specificity afforded by using human-derived P. jirovecii Msg peptides has been shown to be useful for serological analysis of human sera. Thus, sera from patients in Yaoundé, the capital city of Cameroon, were analyzed for anti- P. jirovecii antibodies by enzyme-linked immunosorbent assay (ELISA) using three recombinant major surface glycoprotein (Msg) peptide fragments, MsgA1, MsgB, and MsgC1. Based on serum recognition of one or more of the three fragments, 82% of the total samples analyzed was positive for antibodies to P. jirovecii Msg, indicating high prevalence of P. jirovecii infection or colonization among Cameroonians. Different Msg fragments appear to be recognized more frequently by sera from different geographic regions of the globe. Antibodies in the Cameroonian serum samples recognized MsgA1 > MsgC1 > MsgB, suggesting that different P. jirovecii strains exist in different parts of the world and/or human populations differ in their response to P. jirovecii. Also, HIV + patients diagnosed with respiratory infections (such as TB and pneumonia) and maintained on trimethoprim/sulfamethoxazol prophylaxis had relatively lower anti-Msg titers. Whether PcP prophylaxis has significant effects on the quality of life among HIV + patients in Cameroon warrants further investigation.

Enhancement of immune response and protection in BALB/c mice immunized with liposomal recombinant major surface glycoprotein of Leishmania (rgp63): The role of bilayer composition

Development of new generation vaccines requires adjuvants to elicit the type and intensity of immune response needed for protection. Liposomes have been shown to be an effective adjuvant formulation. In this study, the role of liposome bilayer composition with different phase transition temperature ( T c) to induce a T helper 1 (Th1) type of immune response and protection against leishmaniasis in BALB/c mice was assessed. Liposome formulations with different bilayer compositions consisting of egg phosphatidylcholine (EPC, T c < 0 °C), dipalmitoylphosphatidylcholine (DPPC, T c 41 °C), or distearoylphosphatidylcholine (DSPC, T c 54 °C) were prepared. All liposomes were contained rgp63 as a recombinant antigen and used to immunize mice subcutaneously 3 times in 3-week intervals. Evaluation of lesion development and splenic parasite burden after challenge with L. major, evaluation of Th1 cytokine (IFN-γ) and Th2 cytokine (IL-4), and titration of IgG isotypes were carried out to assess the type of generated immune response and extent of protection. The results indicated the generated immune response in mice was influenced by the bilayer composition of liposomes, so that mice immunized with liposomes consisting of EPC induced a Th2 type of immune response while liposome consisting of DPPC or DSPC induced Th1 type of immune response. It seems that liposomes prepared with higher Tm phospholipids are suitable formulation to induce Th1 type of immune response and protection, and so might be used for further investigations to develop an effective vaccine against leishmaniasis.

The role of liposome charge on immune response generated in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63)

Liposomes as a lipid-based system have been shown to be an effective adjuvant formulation. In this study, the role of liposome charge in induction of a Th1 type of immune response and protection against leishmaniasis in BALB/c mice was studied. Liposomes containing rgp63 were prepared by Dehydration-Rehydration Vesicle (DRV) method. Neutral liposomes consisted of dipalmitoylphosphatidylcholine and cholesterol. Positively and negatively charged liposomes were prepared by adding dimethyldioctadecylammonium bromide (DDAB) or dicetyl phosphate (DCP) to the neutral liposome formulation, respectively. Female BALB/c mice were immunized subcutaneously with negatively, positively charged or neutral liposomes encapsulated with rgp63, rgp63 in soluble form or PBS, three times in 3 week intervals. The extent of protection and type of immune response generated were studied in different groups of mice. The group of mice immunized with rgp63 encapsulated in neutral liposomes showed a significantly ( P < 0.01) smaller footpad swelling upon challenge with Leishmania major compared with positively or negatively charged liposomes. The mice immunized with neutral liposomes also showed a significantly ( P < 0.01) the lowest splenic parasite burden, the highest IgG2a/IgG1 ratio and IFN-γ production and the lowest IL-4 level compared to the other groups. The results indicated that a Th1 type of immune response was induced in mice immunized with neutral liposomes more efficiently than positively charged liposomes and conversely negatively charged liposomes induced a Th2 type of immune response.

The role of CpG ODN in enhancement of immune response and protection in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63) encapsulated in cationic liposome

CpG oligodeoxynucleotides (CpG ODN) are known to be a potent immunoadjuvant for a wide range of antigens. The aim of this study was to evaluate the role of CpG ODN co-encapsulated with rgp63 antigen in cationic liposomes (Lip-rgp63-CpG ODN) in immune response enhancement and protection in BALB/c mice against leishmaniasis. Lip-rgp63-CpG ODN prepared by using dehydration-rehydration vesicle (DRV) method significantly inhibited (P<0.001) Leishmania major infection in mice measured by footpad swelling compared to Lip-rgp63, rgp63 alone, rgp63 plus CpG ODN, PBS or control liposomes. The mice immunized with Lip-rgp63-CpG ODN also showed the lowest spleen parasite burden, highest IgG2a/IgG1 ratio and IFN-gamma production and the lowest IL-4 production compared to the other groups. The results indicate that co-encapsulation of CpG ODN in liposomes improves the immunogenicity of Leishmania antigen.

Immune response and protection assay of recombinant major surface glycoprotein of Leishmania (rgp63) reconstituted with liposomes in BALB/c mice

In this study the ability of recombinant gp63 entrapped in liposomes to induce immune response and protection against L. major infection in susceptible BALB/c mice was studied. Liposomes containing rgp63 (Lip-rgp63) were prepared from egg lecithin and cholesterol using detergent solubilization method. Immunization of BALB/c mice with rgp63 alone conferred a partial protection while entrapment of rgp63 in liposomes significantly increased the rate of protection ( P < 0.05). The parasite burden of spleen in mice challenged with L. major was significantly ( p < 0.001) lower in group of mice immunized with rgp63 alone or Lip-rgp63, however, the least parasite burden was seen in Lip-rgp63 group. Both rgp63 alone and Lip-rgp63 elicited significant delayed-type hypersensitivity (DTH) response compared to controls ( p < 0.01), however, the DTH response of PBS-rgp63 was less than the Lip-rgp63. Titration of anti- Leishmania IgG isotypes (IgG2a/IgG1) showed a preferential Th1 type of immune response only in mice immunized with Lip-rgp63. The results indicate that liposomes might be used as a suitable immunoadjuvant for development of Leishmania vaccine.

Cytokine responses to the native and recombinant forms of the major surface glycoprotein of Pneumocystis carinii.

Pneumocystis carinii is a major opportunistic pathogen and leading cause of morbidity in patients with AIDS. The major surface glycoprotein (MSG) of P. carinii, represented by a family of related proteins encoded by unique genes, is highly immunogenic and contains T cell-protective epitopes. We undertook the present study to define the CD4 T helper (Th) response by cytokine secretion to native MSG and a recombinant form of the protein, MSG-B. Spleen cells were collected from Lewis rats and restimulated with both native MSG and MSG-B. Within 24 h, the CD4 cells secreted high levels of interferon-gamma (IFN-gamma) in response to both types of antigen, indicative of a Th1 response; however, after 72h of incubation, only the native MSG stimulated secretion of IL-4 (Th2 response) from the cells. We then investigated whether the presence of IL-4 could alter the predominant Th1 phenotype by the CD4 cells in response to MSG and MSG-B. Cells cultured with native MSG and IL-4 produced low levels of IFN-gamma and elevated levels of IL-4. Interestingly, cells incubated with MSG-B and IL-4 reduced production of IFN-gamma, but were not stimulated to produce increased levels of IL-4. The presence of anti-IFN-gamma antibody in the MSG- or MSG-B-stimulated cultures did not effect the expression of IFN-gamma mRNA, suggesting that the generation of Th1 cells in response to MSG or MSG-B was not dependent on IFN-gamma. We conclude that native MSG, which contains multiple forms of this antigen, and recombinant MSG elicit different cytokine responses in vitro. These data are not only important to studies of MSG, but may also be relevant to the role of MSG in the immunopathogenesis of P. carinii infection in vivo.

Quantitation of anti-Pneumocystis jiroveci antibodies in healthy persons and immunocompromised patients.

To facilitate studies of the epidemiology of Pneumocystis jiroveci infection in both healthy persons and immunocompromised patients, we developed a quantitative ELISA with recombinant major surface glycoprotein (MSG) fragment MSG-14, a P. jiroveci-specific protein that includes a highly conserved region of the MSG protein family. By immunoblot, all samples reacted with the carboxyl portion of MSG-14; by ELISA, immunocompromised patients with Pneumocystis pneumonia (PCP) who were immunocompromised for reasons other than AIDS had higher antibody levels than did either patients with AIDS with PCP (P=.01) or healthy persons (P=.005). Longitudinal observations of 8 patients with AIDS showed no correlation between time of diagnosis of Pneumocystis infection and change in antibody levels. Eleven percent (4/35) of healthy persons demonstrated a >4-fold change in antibody titers during 1 year of observation. This ELISA assay allows quantitation of anti-P. jiroveci antibodies in human serum samples and should be useful in better understanding the epidemiology of P. jiroveci infection in humans.

The Pneumocystis carinii major surface glycoprotein (MSG): its potential involvement in the pathophysiology of pneumocystosis.

The major antigens of rat Pneumocystis carinii are 45, 60 and 116 kDa, and those of human P. carinii 20, 40, 66, and 95 kDa [1]. The predominant band on acrylamide gels is a surface antigen, which varies in size (95–140 kDa) depending on the host and method of preparation. This antigen is referred to by a variety of names, including gpA, gp95, gp116, gp120 and MSG. In the following the latter term will be used for this antigen. MSG is encoded by a family of approximately 100 genes, each of which is responsible for the expression of a different isoform of MSG [2, 3]. Human MSG is 95 kDa, slightly smaller than the corresponding rat antigen [4,5]. Polyclonal and monoclonal antibodies directed against rat and human P. carinii MSG show that species-specific as well as antigenic determinants are shared and that individual isolates of human and rat P. carinii are antigenically different [4–8]. Biochemical studies indicate that MSG is an acidic glycoprotein composed of a protein core and a carbohydrate portion. N -Linked carbohydrates rich in mannose, glucose, and N -acetylglucosamine residues represent one tenth of the MSG mass [4,9]. Expression of recombinant MSG has largely been unsuccessful. Rat P. carinii MSG has been recombinantly expressed using the Escherichia coli or baculovirus expression system, but differs in glycosylation from native MSG [10]. The difficulty of expressing human recombinant MSG is believed to rely on critical upstream conserved sequences not yet understood. Native MSG preparations from all species are obtained by solubilization of P. carinii organisms with zymolyase and purification utilizing high performance liquid chromatography [11]. Zymolyase hydrolyzes linear glucose polymers with β-1,3-linkages [12]. Studies of the cyst wall have demonstrated that zymolyase treatment removes …

Characterization of major surface glycoprotein genes of human Pneumocystis carinii and high-level expression of a conserved region.

To facilitate studies of Pneumocystis carinii infection in humans, we undertook to better characterize and to express the major surface glycoprotein (MSG) of human P. carinii, an important protein in host-pathogen interactions. Seven MSG genes were cloned from a single isolate by PCR or genomic library screening and were sequenced. The predicted proteins, like rat MSGs, were closely related but unique variants, with a high level of conservation among cysteine residues. A conserved immunodominant region (of approximately 100 amino acids) near the carboxy terminus was expressed at high levels in Escherichia coli and used in Western blot studies. All 49 of the serum samples, which were taken from healthy controls as well as from patients with and without P. carinii pneumonia, were reactive with this peptide by Western blotting, supporting the hypothesis that most adult humans have been infected with P. carinii at some point. This recombinant MSG fragment, which is the first human P. carinii antigen available in large quantities, may be a useful reagent for investigating the epidemiology of P. carinii infection in humans.

Multi-Gene Family of Major Surface Glycoproteins of Pneumocystis carinii: Full-Size cDNA Cloning and Expression

The major surface glycoprotein (MSG) of Pneumocystis carinii plays a crucial role in the fatal pneumonia caused by this organism in AIDS patients. A cDNA encoding a full-length MSG polypeptide was isolated from a lambda library of rat-derived P. carinii cDNAs. The deduced MSG, referred to as the MSG5 subtype, is a 120,765-Da protein composed of 1,076 amino acids and contains an anchoring hydrophobic sequence at the C-terminus of the protein. Sequence analyses of cloned MSG-cDNAs revealed an MSG-gene family with approximately 70% protein sequence identity between subtypes. P. carinii karyotype hybridization analyses indicated that the MSG gene family members are scattered throughout most of the P. carinii chromosomes. These recombinant MSG proteins reacted with the antiserum from P. carinii-infected rats, as expected, and antiserum generated against P. carinii-infected mice, indicating the existence of common determinants in MSG polypeptides. The family of MSG proteins is rich in cysteine residues and these cysteines are highly conserved in all MSG subtypes regardless of species specificity, suggesting the structural and/or functional importance of these cysteines. The pathobiological significance of the MSG gene family and its sequence diversity in P. carinii is discussed.