Host protein glycosylation in nucleic acid vaccines as a potential hurdle in vaccine design for nonviral pathogens.

Nucleic acid vaccines introduce the genetic materials encoding antigenic proteins into host cells. If these proteins are directed into the secretory pathway with a signal/leader sequence, they will be exposed to the host's glycosylation machinery, and, if their amino acid sequences contain consensus sequons for N-linked glycosylation, they may become glycosylated. The presence of host glycans on the proteins of microbial origin may prevent a strong protective immune response either through hindering access to key epitopes by lymphocytes or through altering immune responses by binding to immunoregulatory glycan-binding receptors on immune cells. Ag85A expressed by Mycobacterium tuberculosis (Mtb) is a bacterial surface protein that is commonly used in nucleic acid vaccines in multiple clinical trials. Here we show that, when Ag85A is expressed in mammalian cells, it is glycosylated, does not induce a strong humoral immune response in mice, and does not activate Ag85A-specific lymphocytes as highly as Ag85A natively expressed by the bacterium. Our study indicates that host glycosylation of the vaccine target can impede its antigenicity and immunogenicity. Glycosylation of the antigenic protein targets therefore must be carefully evaluated in designing nucleic acid vaccines.

Identification and characterization of the Streptococcus pneumoniae type 3 capsule-specific glycoside hydrolase of Paenibacillus species 32352.

Bacillus circulans Jordan 32352 was isolated from decaying organic matter in the New Jersey soil in the early 1930s. This soil-dwelling bacterium produced an enzyme capable of degrading the type 3 capsular polysaccharide (Pn3P) of Streptococcus pneumoniae (Spn). Early reports of this enzyme, Pn3Pase, demonstrated its inducibility by, and specificity for Pn3P. We set out to identify and clone this enzyme for its recombinant expression and characterization. We first sequenced the genome of this bacterial species, and reclassified the Pn3Pase producing bacterium as Paenibacillus species 32352. We identified the putative protein of Pn3Pase through mass spectrometry-based proteomics and cloned the gene for recombinant expression. We then characterized the oligosaccharide products generated upon the enzymatic depolymerization of Pn3P. Sequence analysis suggests that this glycoside hydrolase belongs to a new carbohydrate-active enzyme GH family. To our knowledge, this is the only enzyme to demonstrate Pn3P depolymerization activity.

Carbohydrates as T-cell antigens with implications in health and disease.

Glycosylation is arguably the most ubiquitous post-translational modification on proteins in microbial and mammalian cells. During the past few years, there has been intensive research demonstrating that carbohydrates, either in pure forms or in conjunction with proteins or lipids, evoke and modulate adaptive immune responses. We now know that carbohydrates can be directly recognized by T cells or participate in T-cell stimulation as components of T-cell epitopes. T-cell recognition of carbohydrate antigens takes place via their presentation by major histocompatibility complex pathways on antigen-presenting cells. In this review, we summarize studies on carbohydrates as T-cell antigens modulating adaptive immune responses. Through discussion of glycan-containing antigens, such as glycoproteins, glycolipids, zwitterionic polysaccharides and carbohydrate-based glycoconjugate vaccines, we will illustrate the key molecular and cellular interactions between carbohydrate antigens and T cells and the implications of these interactions in health and disease.

Glycosylation as a key parameter in the design of nucleic acid vaccines.

Vaccine-induced immunity is expected to target the native antigens expressed by the pathogens. Therefore, it is highly important to generate vaccine antigens that are immunologically indistinguishable from the native antigens. Nucleic acid vaccines, comprised of DNA, mRNA, or recombinant viral vector vaccines, introduce the genetic material encoding the antigenic protein for the host to express. Because these proteins will undergo host posttranslational modifications, host glycosylation can potentially alter the structure and immunological efficacy of the antigen. In this review, we discuss the potential impact of host protein glycosylation on the immune responses generated by nucleic acid vaccines against bacterial and viral pathogens.

Modulation of immunosuppressant drug treatment to improve SARS-CoV-2 vaccine efficacy in mice.

The COVID-19 pandemic dramatically demonstrated the need for improved vaccination strategies and therapeutic responses to combat infectious diseases. However, the efficacy of vaccines has not yet been demonstrated in combination with commonly used immunosuppressive drug regimens. We sought to determine how common pharmaceutical drugs used in autoimmune disorders can alter immune responses to the SARS-CoV-2 spike protein vaccination. We treated mice with five immunosuppressant drugs (cyclophosphamide, leflunomide, methotrexate, methylprednisolone, and mycophenolate mofetil), each with various mechanisms of action prior to and following immunization with SARS-CoV-2 spike protein. We assessed the functionality of antibody responses to spike protein and compared immune cell populations in mice that received no treatment with those that received continuous or temporarily suspended immune suppressive therapy. All tested immunosuppressants significantly reduced the antibody titers in serum and functional antibody response against SARS-CoV-2 spike protein in immunized mice. Temporarily halting selected immunosuppressants (methylprednisolone and methotrexate, but not cyclophosphamide) improved antibody responses significantly. Through proof-of-principle experiments utilizing a mouse model, we demonstrated that immune suppression in autoimmune disorders through pharmaceutical treatments may impair vaccine response to SARS-CoV-2, and temporary suspension of immunosuppressant treatment may be necessary to mount an effective antibody vaccine response. This work provides feasibility for future clinical assessment of the impact of immunosuppressants on vaccine efficacy in humans.

A Structural Model for the Ligand Binding of Pneumococcal Serotype 3 Capsular Polysaccharide-Specific Protective Antibodies.

Capsular polysaccharides (CPSs) are major virulence factors that decorate the surfaces of many human bacterial pathogens. In their pure form or as glycoconjugate vaccines, CPSs are extensively used in vaccines deployed in clinical practice worldwide. However, our understanding of the structural requirements for interactions between CPSs and antibodies is limited. A longstanding model based on comprehensive observations of antibody repertoires binding to CPSs is that antibodies expressing heavy chain variable gene family 3 (VH3) predominate in these binding interactions in humans and VH3 homologs in mice. Toward understanding this highly conserved interaction, we generated a panel of mouse monoclonal antibodies (MAb) against Streptococcus pneumoniae serotype 3 CPS, determined an X-ray crystal structure of a protective MAb in complex with a hexasaccharide derived from enzymatic hydrolysis of the polysaccharide, and elucidated the structural requirements for this binding interaction. The crystal structure revealed a binding pocket containing aromatic side chains, suggesting the importance of hydrophobicity in the interaction. Through mutational analysis, we determined the amino acids that are critical in carbohydrate binding. Through elucidating the structural and functional properties of a panel of murine MAbs, we offer an explanation for the predominant use of the human VH3 gene family in antibodies against CPSs with implications in knowledge-based vaccine design. IMPORTANCE Infectious diseases caused by pathogenic bacteria are a major threat to human health. Capsular polysaccharides (CPSs) of many pathogenic bacteria have been used as the main components of glycoconjugate vaccines against bacterial diseases in clinical practice worldwide, with various degrees of success. Immunization with a glycoconjugate vaccine elicits T cell help for B cells that produce IgG antibodies to the CPS. Thus, it is important to develop an in-depth understanding of the interactions of carbohydrate epitopes with the antibodies. Structural characterization of the ligand binding of polysaccharide-specific antibodies laid out in this study may have fundamental biological implications for our comprehension of how the humoral immune system recognizes polysaccharide antigens, and in future knowledge-based vaccine design.

Glycopeptide epitope facilitates HIV-1 envelope specific humoral immune responses by eliciting T cell help.

The inherent molecular complexity of human pathogens requires that mammals evolved an adaptive immune system equipped to handle presentation of non-conventional MHC ligands derived from disease-causing agents, such as HIV-1 envelope (Env) glycoprotein. Here, we report that a CD4+ T cell repertoire recognizes a glycopeptide epitope on gp120 presented by MHCII pathway. This glycopeptide is strongly immunogenic in eliciting glycan-dependent cellular and humoral immune responses. The glycopeptide specific CD4+ T cells display a prominent feature of Th2 and Th17 differentiation and exert high efficacy and potency to help Env trimer humoral immune responses. Glycopeptide-induced CD4+ T cell response prior to Env trimer immunization elicits neutralizing antibody development and production of antibodies facilitating uptake of immunogens by antigen-presenting cells. Our identification of gp120 glycopeptide-induced, T cell-specific immune responses offers a foundation for developing future knowledge-based vaccines that elicit strong and long-lasting protective immune responses against HIV-1 infection.

DEK protein level is a biomarker of CD138positive normal and malignant plasma cells.

Overexpression of DEK oncogene is associated with increased proliferation of carcinoma cells and it is observed in several solid tumors due to the amplification of the 6p22.3 chromosomal region where DEK locates. Although the same chromosomal amplification occurs in multiple myeloma (MM), a plasma cell neoplasm, whether the expression and the copy number of the DEK gene are affected in MM remains elusive. We show that despite the increased copy number in CD138positive MM cells (4 out of 41 MM samples), DEK mRNA expression was down-regulated compared with that in CD138negative bone marrow (BM) cells of the same patients (P<0.0001). DEK protein was not detectable by immunohistochemistry (IHC) in CD138positive normal plasma cells or in malignant plasma cells of MM patients (n = 56) whereas it was widely expressed in normal and neoplastic B-cells. Stable knockdown or overexpression of DEK in CD138positive MM cell lines did not affect the proliferation and viability of the cells profoundly in the presence or absence of chemotherapeutic agent melphalan whereas knockdown of DEK moderately but significantly increased the expression level of CD138 (p<0.01). Decreased DEK expression in plasma cells suggests a potential role of this gene in plasma cell development and lack of detectable DEK protein by IHC could be used as a biomarker for normal and malignant plasma cells.