Another important requirement for inducing T cell responses is that there are T cells available that can recognize the peptide. Tan et al. Next to precursor frequencies, binding affinity of peptides to MHC is also a predictor of immunogenicity as has been shown in peripheral blood lymphocytes of acute HBV patients Some groups have shown that it is possible to enhance peptides by increasing binding affinity of the peptide to the MHC molecule 51 , These enhanced peptides might induce a T cell response to conserved, but otherwise too low affinity epitopes.
Another important consideration when vaccinating with short peptides is HLA-specificity. Since peptides of 8—11 amino acids long bind directly into the MHC class I binding groove the peptide has to match the HLA type of the vaccinated individual. To overcome the need for individualized vaccination, Tan et al. HLA-A2 transgenic mice vaccinated with this multi-HLA peptide vaccine, showed a reduction of virus in the lungs and increased survival following influenza infection, compared with mock vaccinated mice, showing that vaccination with peptides can positively influence disease outcome Zhang et al.
Therefore, whole proteins could not be processed through the cross-presentation pathway. Rosalia et al. Soluble protein antigen ended up mostly in the endolysosomes, while long peptides seemed to be more efficiently internalized by DCs leading to a faster intracellular routing. In line with these findings, recent research on peptide vaccination is mainly directed to improving antigen presentation of the peptides of choice, by choosing the right form in which the peptides are presented. Rosario et al.
To induce an effective response against viral infections, there are several requirements that should be met.
One important requirement is that there is a sufficient number of T cells available to kill virus-infected cells. The need for an appropriate magnitude of T cells in order to clear virus was elegantly shown by Thimme et al. Furthermore, an increased breadth of T cell responses can be beneficial. In parallel with these findings, vaccination of mice with a vaccine containing multiple epitopes, were more effective in generating a response to influenza infection than vaccination with single epitopes These findings indicate that a broad response is more effective than a response dedicated to only one peptide.
Another advantage of induction of a broad response is that small mutations of the virus will not lead to escape of the virus from the immune response. Next to a broad response, T cell responses of high avidity also contribute to an antiviral response.
Ex vivo screening of T cell responses in HIV-infected patients showed that controllers reacted to lower antigen concentrations compared to non-controllers, indicating that controllers have T cell responses of higher functional avidity and that this higher avidity is advantageous A fourth requirement is that an effective antiviral response should be of proper functionality to enable control or clearance of the virus. CDa is an indicator of cytotoxic functions such as the production of granzymes and perforins.
Together, these cytokines, chemokines, granzymes, and perforins enable control or clearance of the virus from the host. As reviewed by Seder et al. However, elevated amounts of inflammatory cytokines can also lead to immunopathology as has been shown in H5N1 influenza A virus infection IL is produced by a wide range of cells, including T cells, macrophages and neutrophils. These feedback loops are a way of the immune system to regulate itself, however viral factors can negatively impact this balance as is illustrated in HCV infection.
Another regulatory mechanism is the upregulation of inhibitory receptors such as PD-1, LAG-3, and CTLA-4, which leads to decreased activation potential of T cells and the activation of inhibitory genes in T cells However, upregulation of these receptors has also been shown to be responsible for the exhaustion of T cells and thereby a diminished response in chronic viral infections Summarizing, an effective antiviral response consists of a broad variety of antigen-specific T cells of sufficient magnitude, affinity, and appropriate polyfunctionality.
Furthermore, these T cells should be capable of performing cytotoxic functions, but should not induce immunopathology. Such a response greatly depends on the way antigen is presented to the T cells, emphasizing the important role APCs play in antiviral responses.
In recent years, multiple strategies were developed to increase the quality of antigen presentation of peptides. One of the strategies, already described above, is the addition of CD4 help. However, more general CD4 helper peptides are available. Another group of universal T helper epitopes are natural tetanus sequences, which are very promiscuous in their capacity to bind to MHC class II, and thereby very efficient in acting as a co-stimulus IL induces Th1-mediated immune responses and inhibits Th2-mediated responses Activation of these TLRs can then lead to the production of inflammatory cytokines.
By covalently coupling TLR-activating lipids to the peptide, resulting in so-called lipopeptides, self-adjuvanting peptides are created. Thereby, lipopeptides can signal through the TLRs to induce DC maturation, leading to enhanced antigen presentation. Jackson et al. Chua et al. The addition of lipopeptide resulted in increased DC maturation at low doses of the vaccine Indeed, lipopeptide vaccination can induce protective CTL responses, as shown by Day et al.
To improve the effectiveness of peptide vaccines, there are several types of adjuvants available, with different effector mechanisms. Some adjuvants induce depot formation; others directly stimulate the immune response through additional signals. In earlier work on peptide vaccination, strong adjuvants were necessary for induction of immunogenicity.
A commonly used adjuvant for peptide vaccination is IFA, which was applied in the first peptide vaccine, or the human equivalent Montanide. Research by den Boer et al. This depot of antigen and adjuvant can lead to chronic inflammation of the site of injection that may persist for a long time. Harris et al. However, the risk with such depots is that the peptide might be present for a long time after vaccination, but the adjuvant might not be, allowing presentation of the peptide without the necessary co-stimulation and with the risk of inducing tolerance Furthermore, although effective in therapeutic vaccination, IFA does lead to the formation of lesions on the site of injection, making it less attractive for use in a preventive vaccine Two clinical trials, one with HIV peptides and another with malaria surface proteins mixed in Montanide, have even been terminated because of these severe adverse events 88 , An alternative for water in oil formulations could be the use of vesicular delivery systems.
Depending on the nature of the delivery system, they provide the possibility to incorporate immune modulators to direct the immune response, protect against degradation of the peptide, directly target the antigen to the place of interest and, finally, actively transport the antigen across the target membrane. Currently, there are several delivery systems available for peptide vaccination, i.
Liposomes consist of a lipid bilayer, in which antigens or other substances can be entrapped in the lumen or the lipid bilayer, depending on traits of the peptide. The lipid bilayer of liposomes can fuse with other bilayers, such as a cell membrane.
Thereby, liposomes can deliver antigens to the cytosol of APCs Liposomes, containing a short CD8 lipopeptide in combination with CpG, were able to induce protection in a murine influenza challenge model However, liposomes cannot induce maturation of DCs without addition of an adjuvant and are therefore not sufficient to induce co-stimulation. To address this problem, several groups are developing modified liposomes to increase targeting to DCs by adding targets for C-type lectin receptors such as glycans or mannose, which are typically expressed on DCs Virosomes, or influenza derived virus-like particles, have similar membrane-fusion capacities as live influenza virus, which allows them to actively fuse with cell membranes and thereby deliver antigens directly into the cytosol of APCs leading to cross-presentation of antigenic peptides Furthermore, they have been shown to induce up regulation of maturation markers on bone marrow-derived DCs, in mouse models 95 , However, as of now, DC maturation capabilities of virosomes have not been shown in human systems.
Thus, liposomes, virosomes, and other delivery systems can successfully be used to deliver antigens to the place of interest. In addition, they can provide the necessary co-stimulation for APCs, either due to their own properties or by adding other adjuvants to the formulation. The first and most successful, peptide-based vaccine that is currently licensed is a therapeutic vaccine against HPV.
Since the success of this therapeutic cancer vaccine, many groups are exploring peptide vaccination for other viral agents. Therapeutic vaccination, with synthetic peptides, of HCV patients not responding to standard treatment, resulted in a decrease in viral RNA as shown in two separate studies.
Klade et al. All patients that were vaccinated intradermally with TLR7 agonist imiquimod as adjuvant, showed a modest decline in viral titers The study by El-Awady et al. However, although these studies provide a proof of concept for peptide vaccination for therapeutic use in HCV infection, the improvements are only minor.
For a preventive peptide vaccine, there are different necessities. First of all, it should target conserved sequences, which could lead to a universal vaccine. Especially in influenza vaccine development, the threat for a new pandemic to occur has boosted research on the development of such a universal vaccine.
The research of Tan et al. Other vaccination strategies, currently in development, include the use of virus-like particles in combination with an antibody-inducing influenza protein such as the relatively conserved M2e protein or lipopeptide in combination with liposomes 92 , 99 , A recent advancement is that there are some peptide-based vaccines against influenza virus infection in Phase I clinical trials, that are able to induce vaccine-specific cellular immunity , First, the objective of vaccination should be taken into consideration.
Vaccines can be largely divided into therapeutic and preventive. Preventive peptide-based vaccines should elicit a robust memory T cell response, since vaccine-induced T cells need to respond rapidly after infection to clear the virus before it causes illness or at least to limit disease burden.
In the case of therapeutic vaccination to chronic infections, the response should be vigorous and elongated and a rapid response is of less importance. Both for therapeutic and preventive vaccines, eliciting this response at the required location is of great value. Peptide-based vaccines for respiratory viruses, for example, might be more effective when administered intranasally, since lung resident immune cells might then be primed more easily However, changing the route of administration is not always sufficient and then adjuvants in the form of delivery vehicles might aid in transporting vaccine components to the right location in order to elicit an efficient T cell response.
There are several factors to take into consideration when designing peptide-based vaccines, such as location of the response, type of response to be induced, and side effects. The contribution of these factors in the design of preventive versus therapeutic vaccines are summarized in the table. Although inducing T cell responses is very important in protection against many pathogens, there are also indications that these T cell responses cause harm.
In the case of HCV infection, a broad and specific T cell response is able to control virus infection However, during chronic viral infection, liver damage occurs, which is assumed to be immune-mediated. In a study by Maini et al. In contrast, Feuth et al. Since a large number of T cells are detected in the liver of patients with liver damage, damage has been proposed to be caused by the recruitment of non-virus-specific T cells Although in humans the mechanism by which immunopathology develops is not clear, it is important to bear in mind that an exaggerated T cell response to infection or vaccination may lead to unwanted immune-mediated damage.
Therefore, vaccine-induced T cell responses should be effective against the virus, without eliciting major side effects. Traits of the host also influence the effectiveness of a vaccine. Therefore, it is important to consider the target group for vaccination.
Figure 6. Materials and Methods Cell lines T. Plasmid construction The glycoprotein expression plasmids were constructed similarly to previously reported [29]. Production of pseudotyped viral particles Recombinant lentiviruses were prepared by transient transfection of T cells using a standard calcium phosphate precipitation protocol [49].
Confocal imaging of GFP-vpr labeled virions GFP-vpr-labeled lentiviral particles were produced as previously described [32]. Virus attachment assays Production of [ 35 S]-methionine-labeled viruses were produced by transfection of T cells as described above. Assays to inhibit pseudotyped virus-mediated infection In dose-response experiments, T. Acknowledgments We thank Dr. References 1. Immunopharmacol Immunotoxicol — View Article Google Scholar 2.
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Abstract Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. Keywords: egress, nuclear envelope, deenvelopment, perinuclear space. Open in a separate window. Table 1. Virus Cells No. Accumulation of gB and gH in the NE. Immunoelectron Microscopic Analyses of Nuclear Herniations. Discussion Herpesviruses egress from host cells is a complex process, especially given that large capsids cross the NE without cell lysis.
Materials and Methods Cells. Supplementary Material Supporting Figures: Click here to view. Acknowledgments We thank Catherine Wright for excellent technical assistance with the EM experiments.
Footnotes The authors declare no conflict of interest. References 1. J Struct Biol. J Virol. Mettenleiter TC. Mettenleiter TC, Minson T. Curr Opin Microbiol. Campadelli-Fiume G, Roizman B. Roizman B, Knipe DM. Herpes Simplex Viruses and Their Replication.
Philadelphia: Lippincott, Willams, and Wilkins; Arch Virol. Spear PG, Longnecker R. Farnsworth A, Johnson DC. Hutchinson L, Johnson DC. Herrmann H, Aebi U. Annu Rev Biochem. J Cell Physiol. J Gen Virol. Lee SK, Longnecker R. Timms BG.
Am J Anat. Plaque formation was significantly delayed with these mutants and the plaques were smaller and more diffuse than those produced by wild type VSV. In addition, cells infected with these mutants produced approximately 5- to fold less infectious virus than cells infected with a similarly recovered VSV encoding the wild-type G protein.
We also show that the mutant viruses were more sensitive to chloroquine inhibition of infection than either wild-type VSV or the mutant ET, which has a fusion phenotype similar to wild-type G protein.
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