Information

Minimum size for a peptide/protein to be immunogenic in human?


What is the minimum size for an (injected) peptide/protein to cause immunogenic response in human? A reference is very helpful, as well.

Thanks in advance


There's a few limiting factors including how proteins are presented that constrains the actual size needed to get peptide presentation. These depend largely on the MHC molecules (HLA in humans). The binding groove in MHC-I actually limits the size of peptides it can load to 8-10 amino acids, 9 being the most common. This is because "pockets" at either end of the binding groove are said to actually grasp the peptide, and it doesn't allow for much longer sequences. MHC-II lacks these grasping pockets, and so it can bind much longer sequences (Figure 1). MHC-II typically binds sequences of 13-25 amino acids.

Figure 1. Difference between the MHC-I and II binding groove.

There are also a given number of requisite anchor residues that complement the binding pocket of the MHC binding groove that mostly display a particular character at the given position (normally polar at a given position, etc.) (Figure 2).

Figure 2. Just a sampling of possibilities across HLA isoforms.

All information and figures courtesy of Parham "The Immune System," 4th ED.

If the presented peptide has met the prerequisities and has been identified by a leukocyte, in theory it should produce an immune response. I think these are some good considerations to make when you're thinking about how small can a peptide be. I'm assuming here that MHC-II would be handling your injected protein through an extracellular pathway (I can't know for sure), and so the smallest literature MHC-II peptide range I've ever seen is 11 amino acids on the low side. There are a number of computational studies out there that attempt to predict MHC affinity, but it's problematic due to the range of sizes MHC-II may bind and there is variability among the binding cores that contain the anchor amino acids.


Immunogen

An immunogen is an antigen or any substance that may be specifically bound by components of the immune system (antibody, lymphocytes). The term antigen arises from its ability to induce generation of antibodies. Despite the fact that all antigens are recognized by specific lymphocytes or by antibodies, not every antigen can evoke an immune response. Those antigens that are capable of inducing an immune response are said to be immunogenic and are called immunogens. [1]

An immunogen is any antigen that is capable of inducing humoral and/or cell-mediated immune response rather than immunological tolerance. This ability is called immunogenicity. Sometimes the term immunogen is used interchangeably with the term antigen. But only an immunogen can evoke an immune response. [2]

Generally, both are substances that are capable of generating antibodies (antigen) or stimulating immune responses (immunogen).

We can define an immunogen as a complete antigen which is composed of the macromolecular carrier and epitopes (determinants) that can induce immune response. [3]

An explicit example is a hapten. Haptens are low-molecular-weight compounds that may be bound by antibodies, but cannot elicit an immune response. Consequently, the haptens themselves are nonimmunogenic and they cannot evoke an immune response until they bind with a larger carrier immunogenic molecule. The hapten-carrier complex, unlike free hapten, can act as an immunogen and can induce an immune response. [4]


Background

Along with sanitation, vaccines are the most effective and economic public health tools for control of infectious disease [1]. However, vaccine development faces a number of challenges, such as overcoming the limited effectiveness of a number of vaccines, the need for frequent vaccine reformulation, as well as a complete lack of vaccines for some diseases. A central goal of vaccination is to generate long lasting and broadly protective immunity against target pathogens, but this goal is hampered by the variability of both the target pathogens and the human immune system [2]. Current practical solutions to the problem include polyvalent vaccines such as those being developed for dengue virus [3] or seasonal vaccine reformulation against influenza [4].

The majority of traditional vaccines provide protection through neutralizing antibodies and T cells alone rarely offer protection and prevention of diseases. However, they participate in reduction, control, and clearance of intracellular pathogens and have been linked with protective immunity against a number of viral pathogens [5-8]. The biggest success of immunological bioinformatics is the development of algorithms for prediction of peptide binding affinity to the human leukocyte antigen (HLA) - one of the rate limiting steps in T cell-based immune response [9]. Although current forms of these algorithms are highly accurate [10-12], the output alone is not enough to inform the selection of epitopes for therapeutic applications. In the conceptual framework for reverse vaccinology, Rino Rappouli described in silico predictions of immune epitopes from biological sequence data as a "naïve approach" when compared with experimental elucidation immunogenic peptides. Many parameters of a good vaccine target conferring efficient, lasting immunity, still remain to be considered after prediction of HLA binding: multiple rate-limiting steps of peptide pre-processing, confirming in vivo expression, considering dynamics of expression in different developmental stages and cellular environments, presence of epitope across pathogen population, response across host population, epitope stability over time, and others [13]. Here, we address the issue of variability by modifying the antigen selection step with a computational method for selecting multiple T cell targets from functionally homologous protein regions.

Traditionally, vaccine targets are selected from conserved regions in the genome of the pathogen in question, with the aim of conferring broad and lasting immunity. The first step is a variability analysis performed by calculating the frequency of nucleotides or amino acids on each position in a multiple sequence alignment (MSA) of homologous genes or proteins [14]. Regions, in which several consecutive residues show high conservation (typically 㺐% conservation is chosen as the threshold), are then further analyzed for immunogenic potential either by computational predictions, experimental testing, or a combination thereof. This systematic exclusion of low frequency variants when using traditional approaches [15-19] represents a major limiting factor, since immunogenic potential does not always correlate with the frequency in the viral population - both rare and common peptides can be immunogenic and valuable in vaccine constructs aiming for broad coverage [20].

Since the human immune system's evolution occurs on a significantly longer time scale than rapidly mutating pathogens [21], high selective pressure causes them to alter expression of some immunogenic antigens faster than the immune system can evolve to keep up with the changes [22]. The HLA binding affinity of a peptide relative to its frequency in a viral or malignant cell population is known as its targeting efficiency (TE). It has been shown that the TE of peptides varies in different organisms, and in some highly variable viruses it tends to be low [20]. Regions of high TE comprise peptides that are highly conserved, most likely owing to the protein's functional importance limiting the capacity of a pathogen to alter the protein while maintaining its fitness [23]. Regions of low TE comprise one or more peptides, potentially all of high HLA binding affinity, but each of them will have a low frequency in the pathogen population. For rapidly mutating viruses, such as RNA viruses [24] the selective pressure exerted on HLA-binding peptides, means that host immunity will often, and in some cases preferentially, target low frequency epitopes [20].

Selecting vaccine targets from protein regions with conserved HLA binding

We propose a novel method and visualization scheme for assessing the stability of protein regions for T cell target discovery, which takes the evolutionary relationship between HLA and pathogen epitopes into consideration. This method is based on analyzing columns of suitably sized sliding windows (from here on termed "blocks") from the rows of sequences in an MSA (Figure ​ (Figure1). 1 ). An MSA of homologous protein sequences can be performed using a number of algorithms [25], and blocks of peptides of a given size (usually 8-11 amino acids long for HLA class I restricted epitopes and 13-25 amino acids long for HLA class II restricted T-cell epitopes) are extracted from each position in the alignment. The number of peptides in each block indicates the diversity of the block, for which Shannon entropy and consensus frequency can be calculated as informative metrics [26].

Extraction of blocks from MSA. Subdivision of an MSA into blocks of peptides, l amino acids in length. In this example, l = 9. Block 1 is highlighted in blue. Moving the sliding window to the right in the MSA in increments of one position will give all blocks blocks in the MSA.

In order to identify potential T cell targets, HLA binding affinities are predicted for all peptides in all blocks. Because blocks are extracted from aligned regions of homologous proteins, it is likely that the peptides within a given block display high sequence homology and the majority show similar HLA binding properties even when sequence variations exist. Similarly, the regions surrounding a block will be of high homology, thus increasing the likelihood that peptides from the same block will be processed and presented on the surface of target cells in a similar fashion [27].

Blocks of one or more peptides that are all predicted to bind to the same HLA alleles with similar affinity are potentially valuable targets for polyvalent vaccine designs. This allows for simultaneous immunization with several epitopes - a necessary tactic against highly mutating viruses in which mutations introducing drug resistance can occur within a single day [28,29]. We previously used a rudimentary version of the block conservation analysis for vaccine target discovery in dengue virus (DENV) [30] and reported a 10-fold larger number of potential CD8 + vaccine target candidates as compared to an earlier benchmark study of DENV vaccine target candidates [31]. We here formalize the approach and present a software implementation. To further demonstrate the utility of block conservation, we performed an analysis of HLA class I epitopes in influenza A H7N9 hemagglutinin (HA). The software is integrated into a freely available web service at http://met-hilab.cbs.dtu.dk/blockcons/.


Results

Vaccines are immunogenic and protective in inbred and outbred mice

The immunogenicity and protective efficacy of combination vaccines against CovR/S WT and MT strains were assessed in the murine model of Strep A skin infection, in outbred and inbred mice. SWISS outbred mice (n = 10) were administered J8-CRM + K4S2-CRM/Alum and BALB/c mice (n = 10) were administered p*17-DT + K4S2-DT/Alum, as three intramuscular injections. Final immunizations were followed by skin challenges with Strep A CovR/S MT 5448AP (emm 1) or WT (pNS1, emm 100) strain. Both J8-CRM + K4S2-CRM/Alum and p*17-DT + K4S2-DT/Alum induced peptide specific IgG titers between 10 4 –10 6 (data not shown) and were efficacious in protecting mice (> 99% reduction in bacterial load) against Strep A induced systemic infection from either the WT or MT Strep A strain (Fig. 1a–d).

Protective efficacy of J8-CRM + K4S2-CRM/Alum against invasive disease following skin challenge. (a,b) Cohorts of SWISS mice (n = 10) were immunized intramuscularly with 0.025 mg/dose of J8-CRM + K4S2-CRM/Alum on days 1, 22 and 43. Post final boost, mice were challenged via skin with a Strep A mutant 5448AP or wild type strain pNS1. Mice were culled on day 7 post challenge and blood samples were plated to determine bacterial load. (c,d) Protective efficacy of p*17-DT + K4S2-DT/alum against invasive disease following skin challenge of BALB/c mice. BALB/c mice (n = 10/group, female, 4–6 weeks old) were immunised intramuscularly with 0.025 mg/dose of p*17-DT + K4S2-DT/Alum on days 0, 21 and 42. Two weeks post final immunization, mice were challenged via the skin with 5448AP. Mice were culled on day 7 post challenge and blood samples were plated to determine bacterial load (colony forming units [cfu]). Individual blood bacterial burden (cfu/mL) (a and c) and group percentage reduction in blood bacterial burden (b and d) are shown. Protection is defined as percent reduction in bacterial burden and is calculated using the Geomean bacterial load in blood of vaccinated and control group (PBS/Alum). Statistical comparisons and graphs generated using Mann–Whitney test were performed using GraphPad Prism (8.1.2). ***p < 0.001, ****p < 0.0001.

A repeat-dose toxicity study in Sprague Dawley rats of two candidate vaccines

Two candidate vaccines, J8-CRM + K4S2-CRM/Alum and p*17-CRM + K4S2-CRM/Alum, and a control article, CRM/Alum, were administered to male and female Sprague–Dawley rats (n = 20, 10/sex/group) on days 1, 22 and 43. Rats received 0.5 mL intramuscular injections (0.25 mL/thigh) as follows: Group 1—0.065 mg CRM/Alum (control) Group 2—0.1 mg J8-CRM + K4S2-CRM/Alum Group 3—0.1 mg p*17-CRM + K4S2-CRM/Alum. The control article, CRM/Alum, contained 0.065 mg of CRM which is the average concentration of CRM contained in either vaccine. J8-CRM + K4S2-CRM/Alum contained 0.05 mg of J8-CRM and 0.05 mg of K4S2-CRM for a net peptide conjugate concentration of 0.1 mg. p*17-CRM + K4S2-CRM/Alum contained 0.05 mg of p*17-CRM and 0.05 mg of K4S2-CRM for a net peptide conjugate concentration of 0.1 mg (Table 1). All study animals were observed throughout treatment until half of the study animals were euthanized and necropsied on study day 57 (Main cohort, 2 weeks after dose 3) the remaining rats were euthanized and necropsied on study day 86 (Recovery cohort, 6 weeks after dose 3).

All animals survived until scheduled sacrifice. No adverse vaccine-related findings were reported in the clinical signs, body weights, food consumption, ophthalmology, gross pathology or macroscopic observations. All microscopic changes observed in vaccine groups were within the spectrum of expected findings post-vaccination and considered non-adverse.

Local irritation assessment (draize scoring)

Minor erythema and edema were observed in all dose groups, after the first and second doses. Following the third dose, an effect on erythema and edema at the injection sites were observed in the female vaccine groups when compared to the females in the CRM/Alum control group. Findings in the male vaccine groups scores compared to the control group were not considered toxicologically relevant.

In the female control group, the onset of erythema occurred 48 h post dose and began to resolve 72 h post dose. In both the female vaccine groups, the peak onset of erythema occurred at 24 h and persisted through to 72 h post dose. A significant increase in erythema (left and right) was observed at 24 and 72 h post dose in the both the vaccine groups. Additionally, at 72 h post dose, an increase in left and right leg edema scores was observed in the female J8-CRM + K4S2-CRM/Alum group, compared to the control group. This was also observed in the female p*17-CRM + K4S2-CRM/Alum group, where a significant increase in left leg edema and an increase (not significant) in right leg edema was observed at 72 h post dose (Fig. 2). These observations were considered non-adverse and no other toxicologically relevant effects on local irritation were noted.

Female Draize scoring after third dose (day 43). Draize scores are treated as replicates (n = 10), for each clinical observation, at a given time point. All four clinical observations (a) left leg erythema (b) left leg edema (c) right leg erythema and (d) right leg edema are shown for each time point. Data for an observation, at a given time point, is displayed as group score mean with SD. Statistical analysis and graphs generated using T test to compare each vaccine group to the CRM/Alum control, at each time point, were performed using GraphPad Prism (8.1.2). *p < 0.05, **p < 0.01.

Clinical pathology

No adverse vaccine-related findings in the clinical chemistry, coagulation or urinalysis were reported throughout the study phases. No effect on hematology parameters were considered vaccine-related, during the dosing phase of the study. At the day-86 sacrifice, a modest increase in white blood cell and lymphocyte counts were reported in both female groups receiving vaccine (5% significant difference from CRM/Alum control group). Mildly elevated monocyte counts were also observed at day 86 in females receiving p*17-CRM + K4S2-CRM/Alum (5% significant difference from CRM/Alum control group). These increases were not considered adverse, but rather a reflection of the immune response induced by immunization.

Immunogenicity and functional assessment of vaccines in rats

There were no peptide-specific antibodies detected in sera samples collected from rats prior to dosing (Fig. 3a,b). There was a robust immune response to the Strep A peptides, J8 and K4S2, in the J8-CRM + K4S2-CRM/Alum main group animals (day 57 sera) that persisted in the recovery group animals (day 86 sera) (Fig. 3a). Additionally, there was a robust immune response to the Strep A peptides, p*17 and K4S2, in the p*17-CRM + K4S2-CRM/Alum main group animals that persisted in the recovery animals (Fig. 3b).

Vaccine immunogenicity and functionality of vaccine-induced antibodies. (a,b) Vaccine peptide specific serum IgG titers induced in Sprague–Dawley rats after vaccination with J8-CRM + K4S2-CRM/Alum or p*17-CRM + K4S2-CRM/Alum. Sprague–Dawley rats were immunized intramuscularly with 0.1 mg/dose of J8-CRM + K4S2-CRM/Alum or p*17-CRM + K4S2-CRM/Alum on days 0, 21 and 42. Rats were euthanized at day 57 and day 86. J8, p*17 and K4S2 specific serum IgG titers (Geomean) are shown for individual rats vaccinated with J8-CRM + K4S2-CRM/Alum (a) or p*17-CRM + K4S2-CRM/Alum (b) euthanized at day 57 (n = 10 5 male, 5 female) or 86 (n = 10 5 male, 5 female). Pre-vaccination (day 0) serum IgG titers of the same rats assessed at day-57 or -87 are also shown. Samples were considered positive when the mean value of the absorbance, of the highest dilution (1:100), was > 3SD above the mean OD of the negative control. (c,d) Strep A surface binding of vaccine induced antibodies. Sera collected from rats vaccinated with J8-CRM + K4S2-CRM/Alum, p*17-CRM + K4S2-CRM/Alum or CRM/Alum in the toxicology study was assessed for direct IgG binding to heat killed pM1 (c) and 5448AP (d). Mean individual titers of sera collected from days-0 (n = 4) and -57 (n = 4) and assayed by ELISA are shown. Statistical analysis and graphs generated using a Mann–Whitney test to compare all groups, were performed using GraphPad Prism (8.1.2) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

The functionality of vaccine-induced antibodies in rats was assessed via antibody recognition of bacterial proteins. Vaccine induced antibodies generated in the toxicology study were assessed for direct recognition of bacteria proteins by ELISA. Sera collected pre-dosing (day-0) and at main necropsy (day-57), from each cohort J8-CRM + K4S2-CRM/Alum, p*17-CRM + K4S2-CRM/Alum and CRM/Alum (n = 4/cohort), were added to wells coated with heat killed, whole cell preparations of Strep A emm 1 strains pM1 (WT) and 5448AP (MT). Direct binding to bacteria proteins was defined by total IgG titers. Overall, significant binding to bacteria proteins was observed in the day-57 titers for all cohorts compared to the day 0 titers. A comparative analysis between the binding of candidate vaccine-induced antibodies and the control CRM/Alum-induced antibodies was also made. No significant difference in the binding capacity of day-57 J8-CRM + K4S2-CRM/Alum and p*17-CRM + K4S2-CRM/Alum-induced antibodies to wild type or mutant bacteria was observed. Binding to WT pM1 by either J8-CRM + K4S2-CRM/Alum or p*17-CRM + K4S2-CRM/Alum vaccine-induced antibodies was significantly higher (p < 0.05) than the CRM/Alum-induced antibodies. Binding to MT 5448AP by p*17-CRM + K4S2-CRM/Alum vaccine-induced antibodies was significantly higher (p < 0.05) than the CRM/Alum-induced antibodies. There was no significant difference in binding to 5448AP between day-57 J8-CRM + K4S2-CRM/Alum and CRM/Alum. Finally, no significant difference in binding was observed between day 0 titers for all cohorts (Fig. 3c,d).

Organ weights

No vaccine-related weight changes were reported in the brain, epididymis, heart, kidney, liver or lungs at the day-57 or -86 necropsies. A minimal or mild increase in the adrenal gland weights in male rats receiving the vaccine candidates, compared to the CRM/Alum control, was observed at the day-57 sacrifice. This was considered a common change in toxicologic pathology. The histopathologic correlate was adrenocortical hypertrophy, which is typically secondary to physiologic stress. No vaccine-related organ weight changes were reported at the day-86 sacrifice.

Histopathological analysis

A list of the organs and tissues retained for histopathological examination is provided in Supplementary Table S1. In summary, microscopic observations in the rats that received vaccine, when compared to the CRM/Alum control rats, were granulomatous inflammation at the right and/or left injection sites and inguinal lymph nodes in males and/or females, and adrenocortical hypertrophy in males. All observations were considered non-adverse.

At day-57 sacrifice, the overall incidence of granulomatous inflammation observed at the right and left injection sites was comparable between the control group and the vaccine groups (Supplementary Table S2). There was a slight increase in the severity of the granulomatous inflammation in both the left and right injection sites of male rats and the right injection site of female rats that received J8-CRM + K4S2-CRM/Alum. Likewise, there was a small increase in severity of the granulomatous inflammation in the left and right injection sites of female rats and the left injection site of male rats that received p*17-CRM + K4S2-CRM/Alum. At day 86, a slight increase in severity of granulomatous inflammation persisted in the right injection site of one of five female rats that received p*17-CRM + K4S2-CRM/Alum. No evidence of persisting granulomatous inflammation was observed in the left injection site. The injection site granulomas contained aggregates of macrophages interspersed with small numbers of lymphocytes and plasma cells typically around the periphery of the macrophage aggregates.

An increase in the severity of granulomatous inflammation in vaccine groups was observed in the inguinal lymph nodes (Supplementary Table S3). At the day-57 sacrifice, the group administered J8-CRM + K4S2-CRM/Alum experienced a slight increase in the severity of granulomatous inflammation of the left and right nodes of female rats and the right nodes of male rats. The overall incidence of this finding was considered similar across the control and vaccine groups. In addition, at the day-86 sacrifice, a small increase in severity was observed in the left inguinal lymph nodes of female rats in both vaccine groups. The increased granulomatous inflammation was not observed in the left inguinal nodes of male rats or the right inguinal nodes of any of the animals. As with the granulomatous inflammation at the injection site, the granulomas in the inguinal lymph nodes, were characterized by the presence of aggregates of macrophages. The cytoplasmic material of the individual macrophages was suggestive of phagocytosed adjuvant.

At the day-57 sacrifice, minimal or mild adrenocortical hypertrophy was reported in male rats in both vaccine groups, compared to the CRM/Alum control group (Supplementary Table S4). No incidence of moderate or marked hypertrophy was seen in any animal. The cytoplasm of the cortical epithelial cells was described as homogenously eosinophilic, whilst the increase in cell size resulted in compression of the proximate blood vessels. The adrenocortical hypertrophy was the histopathologic correlate for the increased adrenal gland weights, a typical stress response observed in toxicity studies 20 . In the context of this study, the observation was considered secondary to physiologic stress and not vaccine-related. There was no report of adrenocortical hypertrophy at the day-86 sacrifice.

Examination of the kidneys, brain and joints

No vaccine-related adverse findings were reported in the kidneys. At both the day-57 and -86 necropsies, there was no evidence of macroscopic damage (lesions) or changes to the organ weight. There was also no effect on urinalysis parameters, including blood and protein, observed after the administration of either vaccine compared to the CRM/Alum control.

A broad spectrum of histopathological findings in the kidneys were reported at both day-57 and -86 necropsies and are outlined in Table 2. These findings were of similar incidence and severity in the CRM/Alum control and vaccine treated groups. The observations were considered to be incidental for the strain and age of the rats and not vaccine-related.

An examination of the brains of all animals found no evidence of macroscopic damage (lesions) or histopathological findings at the day-57 necropsy and no evidence of a change to the organ weight at day 57 or 86. At day 86, a single male rat administered p*17-CRM + K4S2-CRM/Alum was found to have mild dilation of a ventricle. No other histological findings related to the brain were reported for day 86.

There was also no report of any inflammation observed in the joints of any animals receiving vaccine.

Examination of the heart

A microscopic examination of the heart found no evidence of valvulitis, however, minimal cardiomyopathy was observed in 11 of 60 animals and mild cardiomyopathy in 1 animal no incidence of moderate or marked myopathy was seen in any animal (Table 3). Statistical analysis (Fisher’s exact test, p < 0.05, GraphPad Prism) of cardiomyopathy observed at day 56 in groups that received vaccine compared to the control article determined no significant difference in the rates observed (J8-CRM + K4S2-CRM/Alum p value 0.9999, p*17-CRM + K4S2-CRM/Alum P value 0.9999). In addition, observations at day 86 in groups that received vaccine compared to control article determined no significant difference in the cardiomyopathy rates observed (J8-CRM + K4S2-CRM/Alum p value 0.0867, p*17-CRM + K4S2-CRM/Alum p value 0.2105). The observation of cardiomyopathy is in-line with other toxicology studies, where spontaneous cardiomyopathy in young Sprague–Dawley rats is a common background finding and has been reported to have up to a 100% incidence, particularly in males 21 . The overall conclusion was that it is an incidental finding not related to the vaccines. This supports our previous findings with J8 in a heart valvulitis model 13 .

Stability of vaccines over 12 months

Stability of the vaccines, for long-term storage at 5 °C ± 3 °C, was assessed at 3-, 6-, and 12-month time points (T3, T6, T12), post manufacturing (Time 0). Stability parameters assessed were appearance, pH, adsorption to Alum (Table 4) and immunogenicity (Fig. 4). The vaccines were shown to be stable for up to 12 months, when stored at 5 °C ± 3 °C, with no significant changes noted to any parameters measured over that time. This included inducing an expected potent immune response in Sprague Dawley rats.

Stability of vaccines. Peptide specific serum IgG titers induced in Sprague–Dawley rats, after vaccination with J8-CRM + K4S2-CRM/Alum or p*17-CRM + K4S2-CRM/Alum stored for 0, 3, 6 and 12 months, were compared. Sprague–Dawley rats were immunized once intramuscularly with 0.1 mg/dose of J8-CRM + K4S2-CRM/Alum or p*17-CRM + K4S2-CRM/Alum. 18 ± 2 days following immunization, rats were bled. J8, p*17and K4S2 specific serum IgG titers are shown. IgG titers in rats (n = 6 3 male, 3 female) vaccinated with J8-CRM + K4S2-CRM/Alum (a) or p*17-CRM + K4S2-CRM/Alum (b) stored for 0 (T0), 3 (T3), 6 (T6) and 12 months (T12). Samples were considered positive when the mean value of the absorbance, of the highest dilution (1:100) was 3SD above the mean OD of the negative control. Statistical analysis using a Mann–Whitney test to compare all groups to the T0 group, were performed using GraphPad Prism (8.1.2). *p < 0.05.


Results and discussion

Design of CHOPS

The Staphylococcal protein CHIPS is one of the most potent inhibitors of C5a-induced inflammatory responses presently known. In contrast to the numerous agents developed to interact directly with the C5aR activation site located inside the receptor core (Proctor et al. 2006 Chen et al. 2010), CHIPS blocks activation by C5a by binding with high affinity to the flexible extra-cellular N-terminal portion of the C5aR (Postma et al. 2005). The interaction surface of CHIPS31–121 with the C5aR comprises

20% of its solvent accessible surface and is not confined to a limited region of the protein. The interactions between CHIPS and the C5aR involve a substantial number of non-sequential amino acids optimally positioned in the inhibitory protein to provide tight binding. A successful mimic of CHIPS should not only include the amino acid residues (or mimics of these) crucial for C5aR binding, but also the amino acids responsible for the proper spatial arrangement dictated by the CHIPS folding topology. Our first approach to build such a structure is to leave out a limited number of residues which do not interact directly with the C5aR, but with the intention to leave the structural integrity of CHIPS31–121 intact. NMR titration studies revealed that two regions of CHIPS31–121 show relatively large perturbations in the backbone and Cβ chemical shifts (Ippel et al. 2009): the first region includes residues 43–61, which comprises part of the α-helix and the subsequent loop connecting strand β1 (Fig. 1a). The second region is composed of residues 95–111 and comprises strands β3 and β4 of CHIPS31–121. The non-interacting portion of CHIPS31–121 comprises strand β2 and the long loop connecting β2 with β3 (Fig. 1a). This portion could potentially be left out by directly connecting strand β1 with β3 via a tight turn. Inspection of the NMR structural models of CHIPS31–121 reveals that residue L65 at the end of strand β1 and residue K95 at the start of strand β3 are proximal and offer an excellent opportunity to link the two fragments interacting with the C5aR together to form one short, contiguous sequence. Several β-hairpin inducing sequences have been described and reviewed in the literature (Blanco et al. 1998 Stotz and Topp 2004). One of the smallest peptide fragments, which induces a β-turn and facilitates the formation of an anti-parallel β-sheet, is the dipeptide D-Pro-Gly (Haque and Gellman 1997). This fragment was chosen to link the N- and C-terminal segments of CHIPS, which interact with the C5aR, together. These segments were chosen to comprise the complete elements of secondary structure as present in native CHIPS (i.e. the α-helix and β-strands β1, β3, and β4) in order to pursue structural integrity. The resulting construct consisted of the CHIPS amino acid sequences T36-L65 and K95-G112 interconnected by D-Pro-Gly (Fig. 1b). A number of residues suggested to be part of discontinuous immunogenic epitopes by Gustafsson et al. (2009) are not present in this construct (designated CHOPS). A model representation of CHOPS based on the structure of native CHIPS31–121 is presented in Fig. 1c.

Affinity of CHOPS for the C5aR N terminus

The affinity of the CHOPS fragment for the C5aR was determined using isothermal titration calorimetry (ITC). We synthesized two peptide mimics of the C5aR N terminus: unsulfated peptide C5aR7–28 representing residues 728 of the C5aR and peptide C5aR7–28S2, the same sequence with tyrosine residues 11 and 14 O-sulfated. We titrated a solution of CHOPS with these peptides and recorded the subsequent heat exchange upon formation of the complex. Two typical ITC experiments are shown in Supplementary Figure 3. Clearly, titration of the doubly sulfated peptide C5aR7–28S2 to CHOPS resulted in a substantial exothermic effect (Supplementary Figure 3a) while no significant response was detected in the ITC experiment with the unsulfated peptide C5aR7–28 (Supplementary Figure 3b). Gratifyingly, the affinity of CHOPS for C5aR7–28S2 was in the micromolar range (K d = 3.6 ± 0.2 μM n = 3). The complete thermodynamic analysis of these ITC data plus the comparison with previous ITC studies of CHIPS31–121 and C5a peptide mimics is compiled in Supplementary Table 1.

NMR spectroscopy

Previous NMR studies revealed that the synthetic peptides C5aR7–28 and C5aR7–28S2, which mimic the N-terminal portion of the C5aR, were very flexible in solution and did not have detectable propensity for any preferred secondary structure. Although there is no detailed structure available for the intact C5aR, it is expected that its free extra-cellular N terminus (residues 1–35) is unstructured as well. The protein CHIPS31–121 does adopt a well-defined conformation with flexible regions at the termini and some disorder in the loop region between the α-helix and strand β1 (Haas et al. 2005). As could be inferred from 15 N relaxation studies this particular loop region adopts an ordered conformation in the complex with C5aR7–28S2 (Ippel et al. 2009). NMR spectra of the free CHOPS construct appear to be typical for a largely unstructured polypeptide chain (Supplementary Figure 4a). 2D NOE spectra of free CHOPS contain predominantly sequential NOEs, but a few long-range contacts could be identified. These non-sequential cross-peaks are indicative for an anti-parallel orientation of strands β1 and β3, which are bridged by the β-hairpin inducing D-Pro-Gly sequence (Fig. 2).

Section of the NOESY spectrum of free CHOPS in solution. Several cross-peak assignments are shown indicative for the presence of a β-hairpin comprising strands β1 and β3

Titration of CHOPS with the unsulfated receptor mimic C5aR7–28 did not result in any changes in the 1 H-spectrum of the latter. In contrast, titration of CHOPS with the sulfated receptor mimic C5aR7–28S2 resulted in increased dispersion of resonance lines, which is characteristic for non-random coil behavior (Supplementary Figure 4b). The complex between CHOPS and C5aR7–28S2 is still flexible and the 1 H-spectra show a high degree of overlap. Nevertheless, we could assign some of the NMR signals as indicated in Supplementary Figure 4b. These new signals are at comparable positions as in spectra of the complex between CHIPS31–121 and C5aR7–28S2, and indicative for the formation of native-like structure. Similar features were observed in 1 H– 13 C HSQC spectra upon titration of C5aR7–28S2 to CHOPS (Fig. 3).

1 H– 13 C HSQC spectra of CHOPS. a Methyl group region of the 1 H– 13 C HSQC spectrum free CHOPS. b Similar spectrum of CHOPS in complex with receptor mimic C5aR7–28S2. The assignments are indicated. Peaks originating from C5aR7–28S2 are shown in italic font

The NOESY spectra of the CHOPS:C5aR7–28S2 complex suffer from severe overlap, but still a limited number of long-range intra-molecular and inter-molecular NOE cross-peaks could be assigned. Inter-molecular NOEs were identified between the aromatic side-chain protons of sulfated tyrosine sY14 of C5aR7–28S2 and the side-chains of T53 (Fig. 4a) and Y108 (Fig. 4c) of CHOPS. H13 of C5aR7–28S2 has an NOE contact with V109 of CHOPS (Fig. 4b). Intra-molecular NOEs were identified between T53 and L49 and between Y97 and V109 (Fig. 4a). Similar peaks were observed in NOESY spectra of the complex between CHIPS31–121 and C5aR7–28S2. The position of these residues in the NMR structure of the CHIPS31–121:C5aR7–28S2 complex is indicated in Fig. 4d, e.

Observed cross-peaks in the NOESY spectrum of the CHOPS:C5aR7–28S2 complex in relation to structural features of the CHIPS31–121:C5aR7–28S2 complex (PDB ID code: 2K3U). a c Different sections of the NOESY spectrum recorded at 288 K of a 1:1 mixture of CHOPS and C5aR7–28S2. Identified long-range intra-molecular and inter-molecular NOE cross-peaks are indicated (normal fonts for residues belonging to CHOPS and italic fonts for residues belonging to C5aR7–28S2). d e Cartoon representation of the structure of the CHIPS31–121:C5aR7–28S2 complex. The side-chains of the residues identified in a c are shown in stick representation. NOE cross-peaks observed in a c are indicated by arrows in the structure

CD spectroscopy

The presence of residual structure in the C5aR mimics and CHOPS was also monitored by CD spectroscopy. The CD spectra of the C5a-receptor mimics C5aR7–28 and C5aR7–28S2, and CHOPS show no structural features apart from a shallow minimum at 200 nm (Fig. 5). The CD spectrum of free CHIPS31–121 comprises a large positive signal around 190 nm and a minimum around 205 nm (Fig. 5). This spectrum does not change significantly upon binding of C5aR7–28S2 (data not shown). Titration of CHOPS with a stoichiometric amount of unsulfated receptor mimic C5aR7–28 resulted in an increase of the CD signal, although the shape of the spectrum did not change (Fig. 5a). Stoichiometric titration of CHOPS with sulfated receptor mimic C5aR7–28S2, on the other hand yielded a clear change of the CD spectrum: an increase in the signal around 190 nm and a shift of the minimum from 200 to 205 nm (Fig. 5b). These changes shift the appearance of the CD spectrum towards that of CHIPS31–121 although at lower intensities.

Circular dichroism spectra (CD) of CHOPS, CHIPS31–121 and C5aR mimics. a CD spectra of CHOPS, C5aR7–28, CHOPS:C5aR7–28, and CHIPS31–121. b CD spectra of CHOPS, C5aR7–28S2, CHOPS:C5aR7–28S2, and CHIPS31–121

In this study we aimed to create a shorter version of the immune evasive protein CHIPS based on the NMR structure of the complex between CHIPS31–121 and C5aR7–28S2. The construct we designed (CHOPS) comprises all portions of CHIPS31–121 important in the interaction with the C5aR. Portions outside the binding region including strand β2 and the connecting loop between β2 and β3 were discarded. This was accomplished by coupling strand β1 and β3 together via a D-Pro-Gly linker segment. The resulting 50-residue long peptide appeared to be largely unfolded apart from some residual structure around the β-hairpin inducing D-Pro-Gly linker sequence. ITC studies revealed that CHOPS binds to the doubly sulfated C5a-receptor mimic C5aR7–28S2 with micromolar affinity (K d = 3.6 ± 0.2 μM). Although the affinity of C5aR7–28S2 to CHOPS is three orders of magnitude lower compared to binding to CHIPS31–121 (K d = 8.4 ± 1.2 nM Ippel et al. 2009), this is a very promising result for a first lead compound. No detectable affinity of CHOPS was observed in the ITC measurements using the unsulfated mimic C5aR7–28. This is consistent with previous measurements of CHIPS31–121 and C5aR7–28, which revealed that the absence of the two sulfate moieties results in an almost 400-fold decrease in affinity.

NMR spectroscopy confirmed the results obtained by ITC. Titration of CHOPS with unsulfated receptor mimic C5aR7–28 resulted in the sum of its constituent 1 H-spectra, while titration of doubly sulfated peptide C5aR7–28S2 yielded a completely different 1 H-spectrum with signals shifted from their random coil position. The latter is indicative for the formation of defined structural elements. The NMR titration experiments using C5aR7–28S2 showed binding in a fast-exchange regime, compatible with the observed micromolar affinity by ITC (Cavanagh et al. 2007). Several characteristic features observed in spectra of the CHIPS31–121:C5aR7–28S2 complex were also present in spectra of CHOPS:C5aR7–28S2. Specific resonances of residues L49, T53, T96, Y97, Y108, V109 and N111 have chemical shifts comparable with their counterparts in the CHIPS31–121:C5aR7–28S2 complex. We also observed NOE cross-peaks between residues sY14, T53, L49, and Y108 and between residues H13, V109, and Y97 in NOESY spectra of CHOPS:C5aR7–28S2. These peaks are reminiscent of NOE contacts observed in spectra of the complex between CHIPS31–121 and C5aR7–28S2 and reveal that CHOPS, in the presence of sulfated peptide C5aR7–28S2, adopts similar structures as native CHIPS31–121.

The structural characteristics of CHOPS, either free in solution or in complex with the C5aR mimics, were also studied by CD spectroscopy. The spectra of the separate peptides (CHOPS, C5aR7–28 or C5aR7–28S2) did not show any significant absorption apart from a shallow minimum around 200 nm. Titration of CHOPS with receptor mimic C5aR7–28 increased the amount of absorption, but not the position of its minimum. Titration of CHOPS with the doubly sulfated receptor mimic C5aR7–28S2 caused an increase in absorption around 190 nm as well as a shift of the absorption minimum from 200 to 205 nm. Although the maximum and minimum intensities are smaller, the overall shape of the CD spectrum of the CHOPS:C5aR7–28S2 complex resembles that of native CHIPS31–121.


What is the maximum length of a peptide and minimum length of a protein?

Also, linking to an article would be nice, I know what wikipedia says.

Interesting question. A peptide is technically any two or more amino acids linked by a peptide bond, so there is no maximum size. All proteins are peptide chains, the largest one in humans is titin, a spring-like muscle protein comprised of 34350 amino acids. Its virtually impossible to work with biochemically as a whole. Probably, there are bigger peptides out there.

Protein can also be used to refer to any peptide chain and in nutrition I believe it refers simply to amino acid content. There is no strictly delineated minimal size.

In terms of usage, protein generally refers to a complete gene product, whatever its size. Protein fragment is used to refer to incomplete gene products greater than 30-40 amino acids in length, and peptide is used to refer to everything smaller.


Methods

Cloning

In order to introduce an arginine followed by a stop codon in T2i88-8-pf [21] site-directed mutagenesis PCR was used. The resulting plasmids were transformed into Escherichia Coli strain DH5α by heat shock. The resulting ampicilin-resistant colonies were screened for the recombinant gene by sequencing after miniprep (Promega, Madison, WI, USA). Plasmids harboring the desired genes were transformed into the Escherichia Coli strain BL21(DE3) expression cells (Novagen, Gibbstown, NJ, USA) and a 20 % glycerol stock of each construct (3HR, 2.5HR and 2HR) was stored at −80 °C.

Protein expression

An overnight culture (50 ml) of BL21(DE3) (Novagen, Gibbstown, NJ, USA) cells containing the expression construct was added to LB (3L) supplemented with ampicillin (Fisher, Pittsburgh, PA, USA, 6 ml, 100 mg/ml). The culture was incubated (190 rpm, 37 °C) until the OD600 reached

0.5–0.6, at which time protein expression was induced (IPTG, Fisher, Pittsburgh, PA, USA, 3 ml, 1 M) and the culture incubated further (190 rpm, 37 °C, 3 h). The cells were isolated by centrifugation (4000g, 12 min), and the pellet was stored at −80 °C.

Protein purification

Purification was done under denaturing conditions (9 M urea). Cell pellet was thawed on ice, resuspended in lysis buffer A which is composed of 9 M Urea, 100 mM NaH2PO4, 10 mM Tris and 10 mM β-mercaptoethanol, pH 8.0 and lysed by sonication (SONICATOR → 3000 Ultrasonic Liquid Processor, cycle of 4 s pulse at 55 % amplitude followed by a 6 s rest repeated for a 10 min period). The insoluble cell debris was cleared by centrifugation (45 min at 30,500g). The supernatant was then incubated with nickel beads (Qiagen, Valencia, CA, USA) for one hour. Possible DNA contamination was removed by a wash with buffer B at pH 8.0: 9 M Urea, 500 mM NaH2PO4, 10 mM Tris, 10 mM imidazole. Protein contaminants were washed from the column using a pH gradient and 10 mM imidazole in the buffers. The first wash was done with lysis buffer A and the second and third washes at pHs 6.3 and 5.9 respectively with a buffer containing 9 M Urea, 100 mM NaH2PO4, 20 mM sodium citrate, 10 mM imidazole and 10 mM β- mercaptoethanol. Elution was done at pH 5.2 and pH 4.5 again with 9 M Urea, 100 mM NaH2PO4, 20 mM sodium citrate, 10 mM imidazole and 10 mM β-mercaptoethanol. Any protein that did not elute at the lower pH washes was eluted with buffers containing high concentration of imidazole (buffer C: 9 M Urea, 100 mM NaH2PO4, 10 mM Tris, 250 mM imidazole, pH 8.0 or buffer D: 9 M Urea, 100 mM NaH2PO4, 10 mM Tris, 500 mM imidazole, pH 8.0).

Protein purity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (15 % gel). Following purification, the denatured monomeric peptides were refolded using one of the following three methods: (1) dialysis performed in a stepwise manner (stepwise refolding) (2) one step dialysis against a buffer with no urea (direct refolding) or (3) concentrated (Amicon Ultra centrifugal filter devices, Millipore, Billerica, MA, USA) and diluted to a buffer with no urea (quick refolding). The proteins were filtered with a 0.1 μm polyvinylidene fluoride membrane filter before and after dialysis (Millipore, Billerica, MA, USA, #SLVV 033 RS).

For all three designs (3HR, 2.5HR and 2HR) the protein concentration was calculated using the absorbance at 280 nm with the extinction coefficient (M −1 cm −1 ) and molecular weight (Daltons) of the protein. The extinction coefficient and molecular weight were obtained with ExPASy’s ProtParam tool (http://ca.expasy.org/tools/protparam.html).

Dynamic light scattering

The hydrodynamic diameter was obtained with a Malvern Zetasizer Nano S equipped with a 633 nm laser using a 3 mm path length quartz suprasil cell. The measurements were done at 25 °C and for each protein five scans were collected.

Transmission electron microscopy

Transmission Electron Microscopy samples were negatively stained with 1 % uranyl acetate (SPI) at a peptide concentration of 50 μg/ml. Electron micrographs were taken with a Philips EM 300 transmission electron microscope at an accelerating voltage of 80 kV. The micrographs were scanned at 600 dpi.

Model building

A starting model of the nanoparticle peptide was built using the crystal structures of the helix-turn-helix motif of the channel-forming domain of colicin E1 [24], the tryptophan zipper [25] and a de novo designed trimeric coiled-coil peptide [26] as templates for the linker, the N-terminal pentameric domain and the C-terminal trimeric domain, respectively (Fig. 1a). The model building (Fig. 1b) was done in silico using the graphics program ‘O’ version 12.0.1 [27].

Molecular dynamics simulation

Eleven different constructs were studied by molecular dynamics (MD) simulations with CHARMM 36b1 [28] installed on a Linux cluster at the Biotech Center of the University of Connecticut. The whole MD simulation procedure is divided into five steps: vacuum minimization, solvation, energy minimization, heating and equilibration, and production dynamics. The peptide molecule was solvated and ions were added to achieve a particular salt concentration. In the energy minimization step, we used the steepest descent (SD) method for 50 steps followed by the adopted basis Newton–Raphson (ABNR) method for 50,000 steps, where each step is 1 fs. After the energy minimization of the whole system, the temperature was raised slowly to 300 K from an initial temperature of 100 K at a rate of 10°/1000 steps to relax the molecule and then to run the equilibration for a total time of 100 ps, including the time to raise the temperature. Production dynamics for 2 ns were run thereafter on the whole system for initial screening. Long 10 ns production dynamics were also performed on selected models to find the best one. To analyze the final models, we have calculated root mean square deviation (RMSD) and radius of gyration (RGYR) with respect to the energy minimized structure using CHARMM algorithm [28].


Introduction

Novel coronaviruses are large enveloped single-stranded RNA viruses (size ranging from

32 kb in length) in the coronaviridae family that cause the common cold, influenza-like illness and more serious acute respiratory illnesses, including pneumonia, exacerbations of underlying lung disease, croup and bronchiolitis [1–3]. The subgroups of coronavirus families are alpha (α), beta (β), gamma (γ) and delta (δ) coronaviruses. In particular, this virus was reported to be a member of the β group of coronaviruses. The novel virus was Wuhan coronavirus or 2019 novel coronavirus (2019-nCoV) by the Chinese [4–7]. The International Committee on Taxonomy of Viruses (ICTV) named the virus SARS-CoV-2 and the disease COVID-19 [8–10]. Globally, to date, there has been 1 death every

358 people in the general population. In addition, 89% of the time, the person who died had one or more underlying medical conditions. A total of

8,256,725 coronavirus cases in approximately

3.4% of reported COVID-19 cases have died according to the COVID-19 outbreak [4–7] as of June 17, 2020, 02:16 GMT. By comparison, seasonal flu generally kills far fewer than 1% of those infected [9], estimated by simply looking at the value of current total deaths plus current total recovered and pairing it with a case total in the past that has the same value, i.e.,

445,959/(445,959 + 4,306,426) = 9% CFR (crude fatality ratio) worldwide [11].

Since the 1930s, when the coronavirus family of viruses was first identified, until now commercially, there have been no successful vaccines or antiviral drugs that have been able to prevent or treat infections for COVID-19, and past and current vaccine development efforts against this disease might be of high value for the development of an effective vaccine for COVID-19 [12–14]. According to the WHO,

41 candidate vaccines are being developed for COVID-19 from different countries as of March 13, 2020 (WHO 15 Mar 2020). Many research teams worldwide have been working on monoclonal and polyclonal antibodies against novel coronaviruses produced in vitro using tissue culture techniques, but few of them have entered into trials. Monoclonal antibodies are generally very rarely used in the treatment of infectious diseases. As polyclonal antibodies are composed of a mixture that represents the natural immune response to an antigen, they are prone to a higher risk since they are not all successful. Second, drugs and small molecules can calm the immune system, but patients become seriously ill when their immune system overreacts and starts causing collateral damage to the body. The US Food and Drug Administration today dated June 4, 2020, announced the following actions taken in its ongoing response effort to the COVID-19 pandemic. Dexmedetomidine hydrochloride in

0.9% sodium chloride injection (ANDA-209307) [15] and hydroxychloroquine and chloroquine to treat COVID-19 [16], but side effects such as irregular heartbeats, dizziness or fainting, require medical attention immediately. Another drug use of remdesivir in treatment of COVID-19 is having current drug with possible mechanism of action and chemistry of remdesivir against viral infection [12–14].

The inhaled virus SARS-nCoV-2 likely binds to epithelial cells in the nasal cavity and starts replicating [17]. Human angiotensin-converting enzyme-2 (hACE2) is the main receptor for cell entry in both SARS-CoV and SARS-nCoV-2 outbreaks [18, 19]. The spike glycoprotein (S) on the surface of coronaviruses is essential for virus entry through binding to the hACE2 receptor and for viral fusion with the host cell [20]. Thus, one could target this interaction site between hACE2 and SARS-nCoV-2 spike protein with antibodies or small drug molecules [21]. In vitro data with SARS-nCoV-2 indicate that ciliated cells are primary cells infected in the conducting airways [22]. At this time, the disease COVID-19 is clinically manifest. Overexpression of hACE2 enhanced the disease severity in a physiologically relevant model for investigating hACE2 as a therapeutic target for antiviral intervention against the COVID-19 pandemic. Without a vaccine, we should not think of herd immunity as a light at the end of the tunnel. A vaccine is the only lifetime way to move directly from susceptibility to immunity, bypassing the pain from becoming infected and possibly dying. Moreover, the aim of this study is to focus on identifying selected fragmented (protein subunit) genetic code of antigenic epitope peptides from each novel coronavirus protein domain such as spike protein (S), membrane glycoprotein (M), envelop protein (E) and nucleocapsid protein (N) of the virus and use that as our vaccine. When the vaccine is injected into the body, muscle cells naturally “amplify” it by producing copies of each antigenic epitope peptide from each protein domain, which the immune system detects as a threat. This trains the body’s immune system to defend against novel coronaviruses to produce specific antibodies, such as IgG, IgA, IgM and IgM, by recognizing all antigenic peptides. Finally, only a solution is vaccine that produces antibodies by our own body’s β-cells to fight off infections by novel coronavirus other than any side effects.


VLPs for vaccination against cancer

A strong cytotoxic T lymphocyte (CTL)-mediated immune response is a major factor in eradicating tumor cells and the ability of VLPs to induce these responses by delivering antigen to the cytosol and activating the MHC class I pathway makes them excellent candidates for development of vaccines in the treatment of cancer. Several important elements of the immune system play a role in targeting cancer cells. First, DC cells must receive sufficient signals to mature and stimulate adaptive immunity. This is important to ensure that the immune system does not become tolerant to the antigen by activating regulatory T cells (Tregs) and suppressing the immune system. Second, most cancer antigens are related or identical to self-antigens. Finally, T cells must be able to overcome the immune-suppressive signals generated by tumor cells. VLPs have features that allow them to address these challenges. In this section, we consider the VLP-based vaccines that have been explored as potential anti-tumor treatments [185].

VLP-based vaccines developed against cervical cancer

Some viral infections can lead to cancer. For example, HPV is one of these viruses, especially the two types HPV16 and HPV18, which cause 70% of cervical cancer cases [186]. The virus also causes other cancers such as anal, head, and throat and genital warts. HPV capsid consists of two important proteins called L1 (major) and L2 (minor) proteins. Two commercial vaccines, Merck’s Gardasil®, and GlaxoSmithKline’s Cervarix® have been developed to prevent HPV-related cancer. The expression systems of these two L1-based VLP vaccines are yeast and insect cells, respectively. However, these two vaccines can only prevent cancer associated with genotypes 16 and 18 (Gardasil also protects against genotypes 11 and 6, which cause benign genital warts), and do not protect against other HPV genotypes. Meanwhile these prophylactic vaccines do not cure infected people. Therefore, researchers are still trying to develop a more efficient vaccine. It has been observed that two tumor-specific antigens called E6 and E7 are expressed in all HPV infected cells and increased in cervical tumor cells [187]. E6 inactivates the P53 by binding to ubiquitin ligase E6AP and E7 degrades the phosphorylated retinoblastoma tumor suppressor pRb [188]. Thus, these two proteins repress two critical tumor suppressor proteins. HPV VLP-based vaccines are mainly L1-based because this protein can form VLP. But because the L1 is not conserved between different types of HPV, the researchers have also focused on L2 based vaccine development [189, 190]. However, the L2 challenge is its inability to form VLP [189, 191]. The concatemers consists of tandem or conserves sequences of several HPV types were designed on the MS2 bacteriophage-based VLP vaccine and the mice immunized with the construct produced high antibody titers. They also protected against different types of HPV (16, 18, 31, 33, 45 and 58) in challenge test, as the latest version of the commercial VLP-based vaccine, Gardasil9 [192]. In another study, the bacteriophage AP205 capsid-based VLP with the surface display of the HPV L2-protein and VAR2CSA placental malaria antigen was developed to protect against two diseases, cervical cancer and PM infection, simultaneously. The results showed that this system can induce humoral immunity to protect mice in challenge against both diseases [193].

VLP-based vaccines developed against Breast cancer

Breast cancer is the most common cancer in women and affects men as well (https://www.who.int/). 20–30% of cases of invasive breast cancer is associated with overexpression of human epidermal growth factor receptor 2 (HER2) that is involved in the proliferation and inhibition of programmed cell death [194, 195]. The induction of passive immunogenicity using monoclonal antibodies has been effective in preventing metastasis and tumor growth, but this method is costly, requires multiple administrations at regular intervals for long-term protection and has undesirable side effects [196, 197]. VLP-based vaccines have been shown to address these challenges. Active vaccination with a VLP-based vaccine derived from Acinetobacter phage AP205 coat protein, with surface displayed HER2 protein, was evaluated in FVB mice which had been transplanted with human HER2-positive breast cancer cells. This showed that vaccination was able to inhibit tumor growth in the mice. This approach induced strong humoral immunity and also overcame immune system tolerance [198]. The product of the SLC7A11 gene, xCT, is a transmembrane protein that is overexpressed in cancer stem cells and is a target in breast cancer therapy. This protein activates Treg cells, reduces glutathione synthesis, and helps to encourage tumor invasion as Treg cells suppress the immune system to promote immune tolerance to the tumor cells [199]. A bacteriophage MS2 VLP-based vaccine that displayed the extracellular loop of xCT transporter on its surface was used to treat mice carrying metastatic breast cancer cells. The treated animals produced high levels of specific antibodies and reduced metastasis [200]. Bolli et al. developed the AX09-0M6 as a VLP-based vaccine platform by displaying of the human xCT extracellular domain (ECD6) on its surface. In vaccinated BALB/c mice the neutralizing IgG2a titer was significantly increased and the growth of breast cancer stem cells, xCT activity and pulmonary metastasis was decreased [201].

VLP-based vaccines developed against pancreatic cancer

The cell surface glycoprotein, Mesothelin (MSLN), is overexpressed in many cancers, including pancreatic cancer, and is seen used as a potential anti-cancer drug target. This glycoprotein is involved in cell adhesion and causes cancer cell masses to attach to mesothelial cells. A VLP-based on SHIV VLPs with murine MSLN displayed on the surface of the particles was used to immunize mice harboring pancreatic tumor cells [202]. Following vaccination, tumor growth was inhibited and 60% of the treated mice survived. The VLP strongly stimulated both specific anti-tumor antibody production and CD8 + T cell immunity. It also prevented self-antigen suppression by inhibiting Treg cells.

An alternative therapeutic target for pancreatic cancer treatment is the transmembrane glycoprotein Trop2, which is also overexpressed in other cancers [203]. This protein has little or no expression in healthy epithelial tissue. A simian immunodeficiency virus (SIV) VLP-based vaccine presenting the Trop2 protein was examined for its efficacy against syngeneic pancreatic cancer in C57BL/6 mice [204]. Vaccinated mice showed reduced tumor growth and the VLP-based vaccine significantly activated CD4 + , CD8 + , and the natural killer cell population. Moreover, the population of Treg and myeloid‐derived suppressor cells in the tumor microenvironment was decreased. Decreased expression of immunosuppressive cytokines such as IL-10 and TGF-β confirmed that the treatment inhibited tumor-suppressing immune signals induced by the tumor cells [204].

VLP-based vaccines developed against melanoma

Melanoma is the cause of 10% of all skin tumors and more than 90% of deaths due to skin cancer [205]. A VLP-based vaccine derived from the plant CPMV was used to assess the capacity of and empty CPMV VLP (eCPMV) to suppress tumor growth. Culture medium containing bone marrow-derived DCs (BMDCs) and primary macrophages derived from C57BL6 mice were treated with eCPMV and after 24 h an increase in the expression of some cytokines, such as IL-1β, IL-6, IL-12p40, Ccl3 (MIP1-α), and TNF-α was observed. Treatment of B16F10 lung melanoma cells with eCPMV altered tumor microenvironment immune cell organization. Tumor-infiltrating neutrophil (TIN) numbers were increased and conversely the immune-suppressing cells such as CD11b− Ly6G+ neutrophils were decreased. The mechanism of action of the vaccine was investigated using null mutant mice lacking neutrophils and cytokines IL-12 and IFNγ. In these mice, vaccination was not effective against the tumor and the vaccine did not show its protective anti-tumor effects. This strongly suggests that the beneficial effect was immune-mediated and that neutrophils and IL-12 and IFNγ played a key role in the antitumor effects [206]. The bacteriophage Qβ-based VLP system coupled with TLR9 ligands, in which the surface of each VLP was loaded with one of the Germline or mutated CTL epitopes of B16F10 were also investigated as a potential therapy with positive results. In vaccinated C57BL/6 mice with a mixture of both types of VLP the population of CD8 + T cells specific for the B16F10 murine melanoma significantly increased and tumor progression was inhibited leading to increased survival of the mice. All three types of VLPs were able to provide a degree of protection but the mixture of the two provided significant protection and prevented the progression and invasion of B16f10 cells changed the tumor microenvironment by increasing Ly6G+ granulocytic cells and decreasing Ly6C+ monocytic population [207]. These results indicate that activation of a CD8 + T cell mediated immune response is essential for vaccine efficacy in cancer prevention. In a study to analyze the importance of adjuvant size in stimulating T cell immunity and to evaluate the immune response in a mice model of melanoma a VLP-based platform derived from CMV and incorporating tetanus toxoid epitope TT830–843 (CMVTT-VLP) was established. The p33 peptide epitopes as a model antigen, derived from Lymphocytic choriomeningitis virus, was displayed on the particle surface and the CuMVTT-p33 VLP vaccine was formulated with micron-sized microcrystalline tyrosine (MCT) adjuvant. Comparison of the results with commercial adjuvants, Alum and B type CpGs showed that the micron-sized adjuvant stimulated CD8 + T cell immunity as much as CpG but more potent than Alum. The VLP-based vaccine showed notable antitumor effects against the B16F10 murine tumor cells [208]. VLP-based vaccines for cancer prevention have opened a new era in vaccine research, and although the results so far look promising, more research is needed to reach a definitive conclusion about the effectiveness of these vaccines. The summary of the VLP-based vaccines against different cancers are listed in Table 2.


Background

Shigella is the causative agent of shigellosis, a severe acute gastrointestinal infection that frequently presents as bloody diarrhea, fever, and severe abdominal pain [1]. In 2016, Shigella was estimated to cause > 250 million cases and > 200,000 deaths globally [2]. Higher income countries experience Shigella infections among travelers, aging populations, deployed military personnel, and men who have sex with men (MSM) [2, 3]. However, the preponderance of the Shigella disease burden is in children aged under 5 years residing in low-middle income countries (LMICs). Infection in this vulnerable group can also result in significant long-term consequences such as severe stunting and impaired cognitive development [2, 4]. The global epidemiology of Shigella is worsened by the emergence and spread of multi- and extensively drug resistant (MDR and XDR) variants, making infections increasingly difficult to treat [5]. The principal method of Shigella prevention has been improvements in water, sanitation, and hygiene (WASH) [6]. However, due to the low infectious dose, the standard of WASH required to break transmission is difficult to attain in many LMICs [7]. Furthermore, recent application of molecular techniques to identify Shigella infections found a severe underestimation of the global Shigella burden [8, 9], highlighting the need for new low-cost prevention techniques.

There is currently no licensed vaccine against Shigella [10]. However, studies in animals and controlled human infection models (CHIMs) have shown that protection through immunization is feasible [11, 12]. Natural disease epidemiology in humans and non-human primate infection studies show complete protection from re-infection with a homologous Shigella species. Long-term homologous protection has been attributed to serotype-specific systemic (serum IgG) and mucosal (IgA) antibody responses [12, 13]. The most immunodominant target of the Shigella IgG and IgA response is the O-antigen component of lipopolysaccharide (LPS) [14, 15]. LPS/O-antigen-specific antibodies elicit protection through antibody-mediated opsonization, phagocytosis, and intracellular cytotoxicity [13]. However, antibodies against Shigella O-antigen are highly specific for the infecting species only [11, 13], and do not provide protection against heterologous Shigella species. Since the Shigella genus consists of four species and > 50 serotypes, a lack of cross-protection against heterologous species and serotypes poses a major challenge for vaccine development [16]. This challenge may be overcome by a vaccine that elicits either a broadly reactive immune response or numerous species-specific responses against the globally dominant Shigella species (i.e., S. flexneri and S. sonnei) [17].

The primary strategy of developing an efficacious Shigella vaccine has been to elicit antibody responses targeting Shigella O-antigen [10]. Live-attenuated vaccines with genetically attenuated Shigella [18,19,20], or the expression of Shigella O-antigen on live-attenuated vectors [21], have been shown to induce good antibody responses against O-antigen. Multivalent killed-vaccines also induce high titers of serum IgG and mucosal IgA targeting Shigella O-antigens and have shown protection in early clinical development [22, 23]. Recombinant forms of Shigella O-antigen have also been pursued as vaccine candidates [10], with a Shigella O-antigen conjugated to a carrier protein [24,25,26], which engages T cell help and produces a longer lasting antibody response to the polysaccharide antigen [27]. Various immunogenic proteins, such as the toxins from other pathogens, have been used as carrier proteins [28, 29]. However, protein antigens from Shigella have not been evaluated as a carrier protein for Shigella O-antigen.

Whole genome sequencing provided the ability to predict and derive novel antigens for use as vaccines, and this approach ultimately gave rise to the meningococcal B vaccine [30,31,32]. Further technological advances in immunology and protein engineering to study the interaction of pathogens with the immune system can aid in reverse engineering of protective immunogens [33,34,35]. Here, we aimed to identify novel immunogenic Shigella antigens that could serve as Shigella vaccine candidates, either alone, or when conjugated to Shigella O-antigen. Therefore, we conducted immunogen prediction using bioinformatic analysis, then created a protein microarray of predicted immunogenic Shigella antigens. These expressed antigens were screened for immunogenicity using polyclonal antibodies from patients who recovered from confirmed Shigella infections, to identify a novel set of proteins which may facilitate the development of novel Shigella vaccines.


Example 8

The following fusion proteins are generated PE313-NES-antigen-K, PE1-252-PE268-313-NES-antigen-K, PE1-252-PE280-313-NES-antigen-K. In addition, the fragment of PE domain Ia (PE1-252) of the fusion protein PE313-antigen-NESK is replaced by RAP1 domain 3 (SEQ ID NO: 8). A2M minimum (SEQ ID NO: 9), HIV-Tat minimum (SEQ ID NO: 10) or HSPs minimum (SEQ ID NO: 11) to generate the fusion proteins RAP1 domain 3-PE253-313-antigen-NESK, A2M-PE253-313-antigen-NESK, Tat-PE253-313-antigen-NESK and HSP-PE253-313-antigen-NESK, RAP1 domain 3-PE268-313-antigen-NESK, A2M-PE268-313-antigen-NESK, Tat-PE268-313-antigen-NESK and HSP-PE268-313-antigen-NESK vaccines, RAP1 domain 3-PE280-313-antigen-NESK, A2M-PE280-313-antigen-NESK, Tat-PE280-313-antigen-NESK and HSP-PE280-313-antigen-NESK, respectively. RAP1 domain 3-PE253-313-NES-antigen-K, A2M-PE253-313-NES-antigen-K, Tat-PE253-313-NES-antigen-K and HSP-PE253-313-NES-antigen-K, RAP1 domain 3-PE268-313-NES-antigen-K. A2M-PE268-313-NES-antigen-K, Tat-PE268-313-NES-antigen-K and HSP-PE268-313-NES-antigen-K vaccines, RAP1 domain 3-PE280-313-NES-antigen-K. A2M-PE280-313-NES-antigen-K, Tat-PE280-313-NES-antigen-K and HSP-PE280-313-NES-antigen-K. The cell mediated immune responses enhanced by these vaccines are examined using similar methods as described above.

Table 1 shows SEQ ID NOs. of peptides used for making various fusion proteins.

In summary, the results have proved that a fusion protein containing an APC-binding domain at the N-terminal end, a translocation domain, followed by an antigen of a pathogen, and then a fusion peptide of NESK at the carboxyl terminal end is an improved design over the PE-fusion protein that is without the fusion peptide of NESK at the carboxyl terminus in terms of enhancing cell-mediated immune response, suppressing tumor growth, and/or increasing the percentage of tumor-free animals.

While embodiments of the present invention have been illustrated and described, various modifications and improvements can be made by persons skilled in the art. It is intended that the present invention is not limited to the particular forms as illustrated, and that all the modifications not departing from the spirit and scope of the present invention are within the scope as defined in the appended claims.

The embodiments and examples were chosen and described in order to explain the principles of the invention and their practical application so as to enable others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its spirit and scope. Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.

Some references, which may include patents, patent applications and various publications, are cited and discussed in the description of this invention. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is “prior art” to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.


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