Particular Type of Antigen-Fusion Polypeptides

Thermally Responsive Polymers Fused to Antigens

–Antigen-Carrier-Elastin-Like Peptide Fusion Peptides: Krebs (US13/990344) discloses fusion proteins of an immunogenic carrier protein containg a thermally-responsive aggregation (TRA) polypeptide to control protein aggregation. At lower temperatures the fusion protein is soluble and easy to purify whereas at higher temperature (aboe about 30C) the thermally responsive peptide moeity induces aggregation of the fusion protein which causes higher antigenicity. Examples include an antigen which is linked to a carrier protein (GroIL) containing a thermally-responsive aggregation polypeptide. The fusion proteins can also contain sequence tags for affinity purificaiton (such as His6 tags) or B and T cell activating sequences to enhance immune response. 

Antigen-PolyQ domain fusion: Illyinski (Vaccine 26 (2008) 3223-3226) disclose stimulating aggregation of an antigenic protein by its fusion to polyQdomain enhanced its antigenic potention. Specifically, the weakly immunogenic model antigen GFP was fused to either long polyQ domain that triggered protein aggregation or short polyQ domain that did not promote aggregation. Fusion with the long polyQ domain fusion proteins strongly enhanced anti-GFP CTL activity compared with the smaller polyQ domain fusion. 

Heat Shock Protein (Hsp) fusions:

Hsp fusion proteins elicit antigen specific CTL resposnes in the absence of adjuvants. Immunization of mice with a soluble fusion protein consisting of an ovalbumin fragment, a well charcterized T cell antigen, covalently linked to mycobacterial Hsp70 induced a strong MHC class I restricted CD8 T cell response. Today Hsps are the object of intense work as a potential means of vaccines to treat cancer and other diseases. Hsps as vaccines are a novel approach to disease prevention. Epitope analysis indicates the presence of multiple B and T cell epitopes in many of these Hsps. They can be used as carriers and vectors. Hsp based vaccines, unlike other recombinant protein based vaccines, stimulate both humoral and cell mediated immune respones.  Sairam (US2010/0196416).

Krebs (US13/990344) teaches an antigen fused to a carrier protein such as GroEL fused at its 3′ end to a thermally responsive polypeptide such as an elastin like peptide. 

Sairam (US2010/0196416) discloses a Hsp, in part or whole, either alone or covalently or non-covalently bound to an antigenic molecule, which when adminsitered elicits specific immunolgogical resposnes in a host. The Hsps are DnaK (Hsp70) or GroEL protein (Hsp60) of S. typhi eitehr alone or in combination with antigenic molecules, as a vaccine to augment the immune resposne against S. typhi in mice.

See also antibody production 

Immunogen dosage and route of administration also affect immune response. A dose may be too small or too large to induce an immune response. An insufficient dose will not stimulate an immune response either because it fails to activate enough lymphocytes or because certain ranges of low doses can actually induce a state oftolerance in some cases. An excessively high dose can also induce tolerance. A single dose of most experimental immunogens will also not induce a strong response. Repeated administration or “boosters” over a period of weeks is required to stimulate a strong response. Experimental immunogens generally are administered parenterally(routes other than the digestive tract) as by intravenous, intradermal, subcutaneous, intramuscular and intraperitoneal routes. Antigen administered intravenously is first carried to the spleen, whereas antigen administered subcutaneously moves first to local lymph nodes.The route of antigen injection determines which immune organs and cell populations will be involved in the response.

Immunization with Multiple Antigens 

A potential vaccine against a variable pathogen such HIV whoucld include a variety of antigens to produce an immune response to the variants of the pathogen to which an individual may become exposed (Maksyutov, US 2006/0153865).

Repetitive, multiple site immunization strategy (RIMMS):

Kilpatrick “Rapid development of affinity matured monoclonal anitobides using RIMMS” Hybridoma, 16(4), 1997, discloses RIMMS immunization of antigen into several subcutaneous sites, proximal to draining lympodes of mice over the course of 7-11 days.

Immunization with plurality of antigens, generally

Sawyer (US2005/0181483) discloses methods for producing monoclonal antibodies by introducing at least one antigen (e.g., multiple antigens) into an animal, recovering antibody producing cells, generating immotalized cell line and screening the supernant against a protein chip to select a monoclonal antibody that binds to the candidate antigen. The animal may be immunised with the antigen(s) via more than one of a number of possible routes (e.g., intrasplenically, intravenously, intraperitoneally, intradermally or subcutaneously). For example, some of the purified antigen(s) may be injected intraperitoenally and the rest subctaneously. 

Stinchcomb (US 2010/0303860) discloses a singel dose vaccine against dengue virus that can include one or more dengue virus serotpye(s). In one embodiment, a subject  is adminsitered at a seaprate site from the first injection, for example next to or in a separate anatomic site. 

Zhang (US2004/0111757) discloses a method for producing a plurality of monoclonal antibody by administering a plurality of antigens or a cellular immunogen made up of a cell population expressing a plurality of antigens to a single animal to induce an immune response agaisnt the plurality of antigens. 

Administration of plurality of antigens, each antigen administered at distinct anatomical site

Deem (US2006/0246081) discloses a method of administering to a subject a plurlity com positions, each composition being adminsitered to a different site of the subject, wherein each site is, or substantially drains to, an anatomically distinct lymph node(s). Each composition includes at least one antigenic molecule having one or more epitopes of the same infectious agent or a strain thereof. The method is advantageous in that it helps against immunodominance where an immune response is elicited against a particular infectious agent, often directed against only a limited number of epitopes or even to a singel epitopoe. Such a narrow immune response to a few immunodominant epitopes offers poor protection against subsequent infection by a mutated form or by different strains of the original infectious agent. 

Chowdhury (US 14/380,128, published as US20150110802)) discloses a method of proudcing a plurality of antibodies in a single animal by immunizing with a plurality of antigens where each antigen species is deliverd to the animal at an anatomically distinct location.

Linking of Antigens on single polypeptide

Multiple antigen peptide system (MAPs):

MPS system is a novel approach for antibody geenration using B eptiopes and T helper epitopes which are linked tandemly on a multi-branched lysine core. ((Yuang, Vaccine, 15(4), pp. 377-386, 1997).

Boosters

Boosters with the Same Antigen:

The use of a prime boost immunization schedule is well known. For example, children typically receive a series of primary immunization up to the age of 15 months (e.g., a DTPa vaccine) and then receive booster doses aged between 4-6 years and beyond (e.g., a Tdap vaccine). Although the priming and boosting vaccines may differ in their precise composition (e.g., the antigen ratios differ in DTPa and Tdap vaccines) antigens used in the two vaccines are typically the same.

Boosters with Different Antigens:

Dormitzer, (US 13/125,526 and 14/219,196) discloses a immunization protocl that uses two or more different antigens which are administered in series or sometimes in combination where the first antigen elicits an efficient germline antibody response and the second antigen elicits an efficient and desired affinity maturation of the antibody response. The first antigen binds to the germline antiboy with greater affinity than to the affinity matured antibody and the second antigen binds to the affinity matured antiboy with greater affinity than to the germline antibody. 

Wysocki (US5,641,488) disclsoes a method for producing antibodies of desired specificity by immunizing an animal with a first immunogen and then with a second immunogen wehre the second immunogen stiulates proliferation of a subpopulation of B cells which have undergone somatic hypermutation to produce antibodies specific to said chosen antigen. In one embodiment, Wysocki disclosing using “Ars” as a first antigen and “Sufl” as the second antigen and that the antibodies which are subsequently generated by immunization with Ars change their specificity to bind “Sulf”. 

Ellenberger (“Recruiting memory B cells with changed antigenic Specificity” J. Immunol., 151, 1993, pp. 5272-5280) also teaches immunization with a first antigen (“Ars-KLH) which recruit and includ somatic mutations in canonical anti-Ars clones haivng acquired new antigenic specificities which were rescued with booster injections with a second related but different antigen (“Sulf-KLH). 

Adjuvants are substances which when mixed with an antigen enhance the immunogenicity of that antigen are often used to boost the immune response. Some adjuvants prolong the persistence of antigen in the immunized animals. For example, when an antigen is mixed with aluminum potassium sulfate (alum), the salt precipitates the antigen resulting in a slower release of antigen from the injection site. Freund’s water in oil adjuvants also function in this way. Freund’s complete adjuvant, which contains heat killed Mycobacteria in the water in oil emulsion, is more potent than the incomplete form because a muramyl dipeptide component of the mycobacterial cell wall activates macrophages, increasing production of interleukin 1 and thus augments the immune response by activating THcells.

Adjuvants areare often used to boost the immune response. Unlike attenuated live vaccines, killed whole organism generally require the addition of an adjuvant to be effective. Some of the features involved in adjuvant selection are the antigen, the animal species to be vaccinated, the route of administration and the likelihood of side effects (Aguilar, Vaccine 25, 2007, 3752-3762). 

Vaccine developers have relied on adjuvants for over 90 years, since the serendipitous discovery of teh immune enhancing effects of aluminum salts. For most of the twentiether centry, aluminum was the only adjuvant included in licensed vaccines. Today, modern adjuvants are designed to overcome twenty-first centruy vaccine challengers. With the introduction of new moleuels, adjuvant formulation now focuses on micro and nanoparticulate platforms, including ano-alum; emulsion or Pickering emulsion; liposomes and combiantion adjuvants sucha s ISCOMS, AS04 and PLG.  (Xinhao Ye, “Increasing vaccine efficacy with novel adjuvants” Genetic Engineering & Biotechnology News, September 2024). 

The FDA has approved six adjuvants for use in human vaccines: aluminum, MF59, AS04, AS03, AS01 and Cpg ODN. Selecting the right adjuvant can elicit an immune response tailored to specific pathogens. (Xinhao Ye, “Increasing vaccine efficacy with novel adjuvants” Genetic Engineering & Biotechnology News, September 2024).

Specific Types of Adjuvants

Aluminum salts (aluminum hydroxide, aluminum phosphate or alum): Some adjuvants prolong the persistence of antigen in the immunized animals. For example, when an antigen is mixed with aluminum potassium sulfate (alum), the salt precipitates the antigen resulting in a slower release of antigen from the injection site. Aluminum adjuvants are widely used in human and veterinary vaccines. They are appropriate adjuvants for vaccines that confer protection by inducing antibodies via the induction of a type 2 immune response, but they do not induce cytotoxic T cell and cell mediated immunity (HogenEsch, Vaccine, 20 (2002), S34-S39. 

Currently used vaccines with aluminum as the adjuvant include Biotrax, Engerix-B, Recombivax HB, Gardasil 9, Covid-19. (Xinhao Ye, “Increasing vaccine efficacy with novel adjuvants” Genetic Engineering & Biotechnology News, September 2024).

–AS04 is a monophoshoryl lipid A (MPL) + aluminum salt approved adjuvant. (Xinhao Ye, “Increasing vaccine efficacy with novel adjuvants” Genetic Engineering & Biotechnology News, September 2024).

Oil in water composed of squalene (MF59): increases antigen-presenting cells, cruitment and activation and promotes anitgen uptake and migration of cells to lymph nodes. 

Tocopherol, squalene and polysorbate 80: promotes local production of cytokines and recuitment of innate cells. It is used in the approved Pandemrix vaccine.

MPL and QS-21, a natural extract of Cilean soapbark tree (AS01): elicits IL1beta and IL-1beta release in antigen presenting cells when co-stimulated with MPLA. It is approved for the Singrix, Mosquirix vaccine.

Cytosine phosphoguanine (CpG ODN): enhances antibody and TH1 responses. I t is approved for the HEPLISAV-B vaccine. 

Escherichia coli Enterotoxin: is one of the most powerful mucosal adjuvants known and has ben used to optimize both mucosal and systemic immune response to an inactivated influenza vaccine for parenteral administration (Couch, Nasal Vaccination, N Engl J Med 2004, 350, 860-1).

Freund’s water in oil adjuvants also function in this way. 

Freund’s complete adjuvant, which contains heat killed Mycobacteria in the water in oil emulsion, is more potent than the incomplete form because a muramyl dipeptide component of the mycobacterial cell wall activates macrophages, increasing production of interleukin 1 and thus augments the immune response by activating THcells.

Heat shock proteins (HSPs): See also HSP fusion peptides in outline.  HSPs have been shown as attractive immunoadjuvants to enhance antigen specific immunity. Vaccination with HSP peptide complexes from autologous tumors or virus infected cells has been shown to eleicity specific antitumor or antivirus immune resposnes. (Zhang J Gene Med 2007, 9: 715-726). 

Monophosphoryl lipd A (MPL): is a derviative of the lipopolysacchrides of the bacterial cell wall and has been approved by the European Medicines Agency (EMEA) as the vaccine FENDRIX 9GlaxoSmithKline), a vaccine against hepatitis B. It was developed by Corixa (Seattle, WA) and is an adjuvant in several other vaccines.

Laser Based Vaccine Adjuvants: 

Novoselova (Photodermatol Phtoimmunol Photomed 2006, 22(1), 33-8) disclose that laser radiation has been widely used during induction of anti-microbial and anti-virus activity and that low power laser irradiation increased IL-2 and Hsp70 production when applied on the thymus projection area.

Onikienko (US 14/255,931, published as US 2014/0335110) discloses using laser radiation as an adjuvant for vaccine. The composition can further comprise HSP70. 

Proteins function as the most potent immunogens, with polysaccharides ranking second. In contrast, lipids and nucleic acids generally do not serve as immunogens unless they are complexed to proteins or polysaccarides. Large insoluble macromolecules generally are more immunogenic than small, soluble ones because the larger molecules are more readily phagocytosed and processed. For example, it is thought that because of the small size of short peptides, peptides rarely encompass sufficeint T cell epitopes in order to provide the necessary cytokine help in the induction of strong immune responses. One strategy used to overcome this problem of immunogenicity include the linking of the peptide to large highly immunogenic protein carriers. The carrier proteins contan large number of peptidic T cell epitopes which are capable of being loaded into MHC molecules, thereby providing bystander T cell help, and/or alternatively the use of strong adjuvants in the vaccine formulation. Examples of these highly immunogenic carriers include the Diptheira and Tetanus toxodis (DT and TT), Keyhold limpet Haemocyanin (KLH) and the pruified protein derivative of Tuberculin (PPD).

Common Antigenic Carrier Proteins

Keyhole limpet hemocyanin (KLH): is the best known carrier protein that is coupled to a synthetic peptide antigen in order to elicit a stronger antibody response. KLH is prone to aggregation which is an important feature that makes KLH a very strong carrier immunogen for commercial antibody production. While KLH is a very good immunogen, it possesses limited solubility and is prone to spontaneous aggregation and precipitation, making it difficult to work with.

The 3 dimensional structure of a compound can influence immunogenicity. Peptides used in a particular vaccine immunogen are often chosen such that they generate an antibody response to the location site of that peptide in the context of the full lenght native protein. Thus, in order to generate antibodies that bind to such chosen locations, the peptide in the immunogen must assume substantially the same shape as it would exist if it was confined by the flanking regions of the full lenght native protein. However, merely conjugating a linear peptide sequence, by conventional chemistry, to a carrier protein rarely ahceives this goal. This is because such an immunogen presents the linear peptide with too much confromational freedom, such thtat the peptide may adopt a loose structure that either is not well recognised by the immune system, or may be entirely different to the conformation adopted by the peptide in the context of the flanking regions of the full lenght native protein. In order to ovecome this conformational freedom problem, it is known to design peptides in a constrained manner, by chemical interactions between distinct amino acid risdues, such that the peptide is held in a curved structure which closely resembles the curve in which the peptide would be held by the flanking sequences in the full lenght native protein (US 5,939,383). To do this it is most common to incorporate two cysteine residues in the peptide sequence between which the desired intramolecular disulphide bridge forms. The cyclised peptide thsu formed is commonly conjugated to a protein carrier to form an immunogen by one of several chemistry methods such as by theio-ether linkages (US 2004/0030106 begin_of_the_skype_highlighting 0030106 FREE  end_of_the_skype_highlighting).

The following are important structural motifs.

(a) Geta-harpin loop structure which often occurs between tow antiparallel beta strands are often relatively easily accessible. As a result, immune reponse are often directed against epitopes present in the beta hairpin sequences.

(b) Cystine-knot (Cys-knot) superfamily. These members have an unusual arrangement of 6 cysteins linked to form a “cystine-knot” conformation with distorted beta-hairpin loops above the knot and a single beta hairpin loop below the knot. The active forms of these proteins are dimers, either homo- or heterodimers. Many members of the Cys-knot family are growth factors.

Adjuvants (see outline)

ProPred (graphical web tool for predicting MHC class I binding regions in antigenic protein sequences). 

Deisgn for binding to MHC Molecules:

It is well established that binding of a peptide to an MHC molecule is a prequisite for activtion of antigen specific T cells. As only certain peptides can bind to a given MHC molecule, the identificaiton of these peptides is one of the bottlenecks in subunit vaccine design. A number of methods have been developed to predict MHC binding peptides. (Sing “ProPred: prediction of HLA-DR binding sites) pp. 1236-1237, vol 17, no. 122001).  

Virus like particles (VLPs): 

Some viral proteins spontaneously assemble into highly repetitive virus like particles (VLPs) and induce strong B cell responses in the absence of adjuvants. Moreover, foreign epitopes inserted into such VLPs induce a similarly strong B cell response. However, the size and nature of epitopes that can be inserted into VLPs is restricted and VLPs containing peptides longer than 20 amino acids often fail to assemble. While peptides genetically fused to either the N or C terminus of VLPs present fewer assembly problems, the immune response obtained against such epitopes are often limited, most likely because the epitopes are not optimally exposed. In addition, such particles may be less stable in vivo.

Hepatits B core antigen (HBcAg) -VLP fusions: Jegerlehner shows that peptides and proteins engineered to contain a free cys can be chemcially coupled to VLPs formed from the hepatits B core antigen (HBcAg) containing a lys in the immuno-dominant region. These antigen-decorated VLPs induced potent and long lived immune responses even against self epitopes in the absence of adjuvants. (Jegerlehner, Vaccine 20 (2002) 3104-3112).

Rabbit haemorrhagic disease virus (RHDV): Peacey show that the capsid protein VP60 of RHDV which spontaneously forms VLP can be genetically modified with various short peptide sequences capable of being presented to and recognised by immune cells. A chemical linker was utilised to covalently conjugate both small peptides and whole protein to the RHDV VLP scaffold. The approach enabled surface conjugation of a substantial range of antigens without the constraints imposed by subunit folding and VLP formation. Attachment of antigen to RHDV VLP conferred the immuno stimulatory properites of the underlying viral shell to the conjugated antigen, and so enable the initiation of both antigen specific humoral and cell mediated immune resposnes.  

Factors which Determine Immunogenicity

Immunogenicity is the ability to induce a humoral and/or cell mediated immune response. Although a substance that induces a specific immune response is usually called an “antigen” the more proper term is an “immunogen.” Antigencity is the ability of an antigen simply to interact specifically with free antibody and/or with antigen binding receptors on lymphocytes. Although all molecules possessing the property of immunogenicity also possess the property of antigenicity, the reverse is not true. Some small molecules called haptens possess the property of antigenicity but are not capable by themselves of inducing a specific immune response.

Factors which influence immunogenicity include the following:

(1) composition and size: (see outline)

(2) Conformation: See right hand panel

(3) foreignness: In order to elicit an immune response, a molecule must be recognized as nonself. The ability to recognize self molecules is thought to arise during development by exposure of immature lymphoctyes to self-components. Any molecule that is not exposed to immature lymphoctyes during this critical period is later recognized as nonself, or foreign, by the immune system.

(4) dose and route: (see outline)

(5) genotype of an immunized animal also influences the type of immune response the animal manifests. MHC gene products which function to present processed antigen to T cells play a central role in this variety. The response of an animal is also influenced by the genes that encode B-cell and T-cell receptors and by genes that encode various proteins involved in immune regulatory mechanisms. Genetic variability in all of these genes affects the immunogenicity of a given macromolecule in different animals.

Companies: Osivax

Nucleic acid based immunization exhibits a number of advantages. For example, the manufacture of nucleic acid based vaccines is straight forward, relatively inexpensive.

DNA Vaccines

Potential advantages: DNA based vaccines are stable for long-term storage. DNA vaccines offer advanatages overy many of vaccines. Since the antigen is produced with mammalin post translational modification, conformation and oligomerization, it is more likely to be similar or identical to the wild type protein produced by viral infection than recombinant or chemically modified proteins. However, this distinction may turn out to be a disadvantage for the application of bacterial antigens, since non-native post translational modification may result in reduced immunogenicity. In addition, viral surface proteins are not highly organized in the absence of matrix proteins.

DNA vaccine can induce humoral and cellular immunity and it also saves labour and time because of the siplicity of oepration (Zhang, J Gene Med 2007 9, 715-726).

Potential disadvantages: DNA based vaccines exhibit a variety of potential safety risks such as induction of anti-DNA antibodies, and potential integration of the transgene into the host genome. This may lead to the inactivation of cellular genes, an uncontrollable long term expression of the transgene, or oncogenesis, and thus, is generally not applicable for tumor-associated antigens with oncogenic potential such as erb-B2.

DNA vaccination in avian species:

Duan (US2005/0208628) dicloses a pcoress for producing antibodies to an antigen in an avian species by delivering a DNA dequence encoding the antigen operatively linke to a promoter capable of directing expression of the antigen in the avian species and recovering antibodies from the avian species. Preferably the avian species being vaccinated is a chicken or quail and the antibodies are recovered from egg yolk. The use of chicken offers a number of advantages such as cost.

RNA Vaccines

Two different strategies have been pursued for immunotherapy with in vitro transcribed RNA (IVT-RNA), which have both been successfully tested in various animal models. Either the RNA is directly injected into the patient by different immunization routes or dendritic cells are transfected with IVT-RNA using conventional transfection methods in vitro and then the transfected dendritic cells are administered to the patient. It has been shown that immunization with RNA transfected dendritic cells induces antigen-specific cytotoxic T-lymphocytes (CTL) in vitro and in vivo. Furthermore, it has been shown that direct injection of naked RNA into the lymph nodes of laboratory animals (intranodal injection) leads to uptake of said RNA primarily by immature dendritic cells, probably by a process called macropinocytosis. It is assumed that the RNA is translated and the expressed protein is presented on the MHC molecules on the surface of the antigen presenting cells to elicit an immune response.

Potential Advantages: Some of the advantages of RNA based immunization are the transient expression and the non-transforming character. Furthermore, RNA does not have to be transported into the nucleus for the transgene to be expressed, and moreover, cannot be integrated into the host genome. Similar to the injection of DNA, the injection of RNA may result in both a cellular as well as a humoral immune response in vivo.

RNA vaccines exhibit important safety features. RNA is more reactive than DNA and less stable but is resistant to ultra-violet radiation. mRNA does not integrate into the host chromosomes. The delivery of mRNA results in faster expression of the antigen of interest and requires fewer copies for expression. mRNA expression is transient, which adds to its safety. mRNA is more effective than DNA for protein production in post mitotic and non-dividing cells because DNA requires translocation through the nuclear member and plasma membrane, while mRNA requires translocation only through the plasma membrane. mRNA is not only a template for translation, but also acts as a ligand for toll-like receptors and is nuclease sensitive; thus it presents less concern for horizontal transmission. In addition, RNA vaccines elegantly integrate adjuvanticity and antigen expression, thereby mimicking relevant aspects of viral infections. This increases their efficacy compared to inactivated vaccines that require the use of adjuvants, simplifying handling and production. RNA can address a range of dedicated immunologic pattern recognition receptors, including TLRs 3, 7 and 8, RIG-1, MDA5, PKR and others that may act synergistically and serve to enhance the induction of antigen specific adaptive B and T cell responses. Importantly, by antigen synthesis in transfected host cells, mRNA vaccines directly introduce antigen into cellular antigen processing and presentation pathways, granting access to MHC molecules and triggering T cell responses, irrespective of the hosts MHC haplotype. This enables the induction of polyclonal T cell responses that may act synergistically with other immune responses, including B cells. (US 11,141,478).

For an infectious disease vacine, what one really wants to see is neutralizing antibodies.The mRNA vaccines are really good at geenrating those particular types of antibodies, adn they are showing that they can generate T cell responses too. (Grinstein, “Vaccine Developers Leverage mRNA and other powerful technologies” Genetic Engineerig & Biotechnology News, May 2024).

Potential Disadvantages: A major disadvantage of RNA based vaccination is the instability of the RNA in vivo, in particular in the cells of the immune system. Degradation of long-chain RNA from the 5′-end is induced in the cell by the so called “decapping” enzyme Dcp2 which cleaves m7GDP from the RNA chain. Thus, it is assumed that the cleavage occurs between the alpha- and beta-phosphate groups of the RNA-cap.

To inhibit the decapping process and thus increase the stability of RNA in vivo, the effect of phosphorothioate-cap-analogs on the stability of said RNA has been studied. It has been shown that the substitution of an oxygen atom for a sulphur atom at the beta-phosphate group of the 5′-cap results in stabilization against Dcp2. The phosphorothioate modification of the RNA 5′-cap has been combined with an “anti-reverse cap analog” (ARCA) modification that inhibits the reverse integration of the cap into an RNA chain. (Sahin, 9,295,717).

Due to its negative charge, mRNA does not easily enter cells, and it can be rapidly degraded by nucleases such as the enzyme RNase. Lipid nanoparticle (LNP) encapsulation, which is used in the current mRNA based COVID vaccines, helps mitigate this problem, as do other methods involving substitution of modified bases or design of the mRNA. Alternatively, physical methods such as electroportion can be employed. This approach has gained traciton in ex vivo adminsitered therapeutic cancer vaccines but is not highly efficient. (Cytiva, “mRNA vaccines and therapeutics: current trends and perspectives” Fe 17, 2025.)

RNA vaccine – cationic lipid nanoparticle:

Huang (US 10,022,435) discloses Aspects nucleic acid vaccines (NAVs) comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, formulated within a cationic lipid nanoparticle.

Against Specific Microbies:

During mRNA vaccine development scientists must identify a target protein form the pathogen of itnerest such as the spike (S) protein in the case of SAR-CoV-2. The target protein must be sufficiently different from porteins in human  cells so the resulting immune response detects and supresses only the pathogen. After target identification, scientists insert the DNA sequence coding for the gene of itnerest(e.g., gene encoding for the S prtoein) into a plasmid, which is amplified in host bacteria (e.g., E. coli), which then udnergoes purification and linerization. Becasue mRNA is highly susceptible to degradation , a lipid nanoparticle (LNP) or other suitable divery system must be developed to package the mRN and deliver it to the cells.

––Coronaviruses are enveloped, positive-sense single-stranded RNA viruses. They have the largest genomes (26-32 kb) among known RNA viruses, and are phylogenetically divided into four genera (α, β, γ, δ), with betacoronaviruses further subdivided into four lineages (A, B, C, D). Coronaviruses infect a wide range of avian and mammalian species, including humans. Of the six known human coronaviruses, four of them (HCoV-OC43, HCoV-229E, HCoV-HKU1 and HCoV-NL63) circulate annually in humans and generally cause mild respiratory diseases, although severity can be greater in infants, elderly, and the immunocompromised. In contrast, the Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus (SARS-CoV), belonging to betacoronavirus lineages C and B, respectively, are highly pathogenic. Both viruses emerged into the human population from animal reservoirs within the last 15 years and caused outbreaks with high case-fatality rates.

Corbett (10,960,070) discloses recombinant coronavirus S ectodomain trimers comprising protomers comprising one or more proline substitution(s) that stabilize the S protein trimer in the prefusion conformation. One class of mutation, comprising one or more (such as two) proline substitutions at or near the boundary between a Heptad Repeat 1 (HR1) and a central helix of the protomers of the coronavirus S ectodomain trimer was found to be surprisingly effective for stabilization of coronavirus S protein trimers in the prefusion conformation. Embodiments of such prefusion-stabilized coronavirus S ectodomain trimers are demonstrated to produce a superior immune response in an animal model compared to corresponding coronavirus S ectodomain trimers that are not stabilized in the prefusion conformation.

–Herpes Simplex Virus (HSV): (US 11,141,478) discloses compositions that include one or more nucleoside modified mRNAs, wherein each of said nucleoside modified mRNAs encodes a herpes Simplex Virus (HSV) glycoprotein or immunogenic fragment thereof, and wherein said nucleoside modified mRNA includes one or more pseudouridine residues. The HSV glycoprotein can include glycoprotein D, C, E, B, H, L or I.

–Zika virus: (US 11,241,490) discloses a composition for inducing an immune response against Zika virus in a subject that includes at least one isolated nucleoside modified RNA encoding at least one Zika virus antigen. In one embodiment, the nucleoside modified RNA includes pseudouridine.

Miles (US 2010/0003285) discloses a vaccine for human hookworm that includes at least one L3 larval stage antigen, at least one adult stage human hookworm antigen and adjuvants.

–SARS: Severe acute respiratory syndrome (SARS) emerged in Guangdong Province in South China in 2002, ultimately spreading to five continents where it caused 8,000 respiratory infections and 800 deaths. The SARS coronavirus (SARS-CoV) was identified as the etiologic agent of SARS in 2003. (US 2016/0376321) discloses methods and compositions related to the SARS-CoV spike protein useful for treatment or prevention of SARS.

–Chagas disease: also known as American Trypanosomiasis is caused by infection with the protozoan parasite Trypanosoma cruzi. It is a leading cause of heart disease in Latin America, with up to 10 million infected people in the Western Hemisphere. The disease burden of Chagas based on disability-adjsuted life years is five times greater than malaria and about one-fifth that of HIV/AIDS in the LAC region. Additionally, the annual economic toll for treatment exceeds 7 billion globablly. Most of the deaths and disability attributed to Chagas diase result form chronic Chagas cardiomyopathy which develops in about 30% of infected individuals years to decades after the initial infection due to cascading effects of parasite induced pathologic changes including inflammation, cardiomyocyte hypertrophy, and fibrosis. (US 2021/0268080) discloses methods for preparing T cruzi antigen presenting dendritic cells which includes transducing the DCs with a vector for the expression of T. cruzi antigens.

See “ways to increase immunogenicity” on outline

See WHO   See History of Vaccines 

Active immunization refers to stimulation of an immune response by challenge with an immunogen. Active immunization occurs after each exposure to an infectious agent (natural immunization) and through exposure to antigens in vaccines. 

Attenuated Viruses :

Companies: Codagenix  Emergex vaccines

Attenuated viruses (i.e., less virulent) pathogens or closely related organisms, was first demonstrated by Edward Jenner in 1796, who vaccinated against smallpox by the administration of a less dangerous cowpox virus. Although a number of live attenuated viruses (e.g., measles, mumps, rubella, varicella, adenovirus, polio, influenza) and bacterial (e.g., bacille Calmette-Guerin (BCG) against tuberculosis) are sucessfully administered for vaccination, there is a risk for the development of serious complications related to a reversion to virulence and infection by the “vaccine” organism, in particular in immunocompromised individuals. The specific design of attenuated viruses is now enabled by recombinant DNA technology through the generation of deletion or mutation variants. unlike isolated proteins, viruses induce prompt and efficient immune responses in the absence of adjuvants. 

Some believe that live, attenuated vacines could play a larger role against many infections, particulalry respiratory infections. While it is clear with some of the mRNA vaccines one gets a good antibody response, it is realtively sort lived. In the influenza setting, one sees most of the infections limited to the winter. This does not seem to be the case with COVID-19 where one sees increases in late spring and early summer. Promoting booster vaccines int he ealry fall if you have lsot most of your immunity covrerage by January or Febraury does not help if you get potentially exposed in the late spring and early summer. 

Components of a pathogen: 

Using components of a pathogen is an alternative approach for vaccination. Isolated components of a pathogen either purified form the pathogen or after expression of a single viral protein (e.g., hepatitis B surface antigen) are administered to induce neutralizing antibodies. Examples include influenza hemagglutinin or neuroaminidase. In the HIV front, recombinant proteins of HIV (gp120 and full lenght gp160) have been evaluated as a means to induce neutralizing antibodies against HIV but with disappointing results. It is well established that the administration of purified proteins is not usually not sufficent to elicit a strong immune response but generally must be given together with helper substances called adjuvants.  

DNA vaccines: (See outline)

Fusion Proteins  (see also drug delivery)

Elastin-like Peptides (ELP) fusions: (Floss, J. Biomedicine and Biotechnology, 2010, article ID 27434) discloses a fusion between elastin-like peptide (ELP) and Ag85B and ESAT-6 which are produced in plants. Mice and piglets immunized with the TBAg-ELP fusion exhibited exhibited a mycobacterial specific immune response with no side effects. The main reactivity of the TBAg-ELP induced antibodies against Ag85B. 

Virus like particles (VLPs):

Companies: Osivax

VLPs are being exploited as vaccines because of both their structural properties and their non-infectious nature. VLPs are supermolecular structures built in a symmetrix manner form many protein molecules of one or more types. Because they lack the viral genome, they are noninfectious. Examples include the capsid proteins of HBV, measles virus, Sindbis virus, rotavirus (US 5071651 and 5374426), foot and mouth disease virus, the retroviral GAG protein (WE 96/30523), the retrotransposon Ty protein p1, the surface protein of HBV (WO 92/11291) and human papilloma virus (WO 98/15631). Renner (WO 00/32227) discloses a (i) molecular scaffold and an (ii) antigen or antigenic determinant. The (i) moelcular scaffold comprises a core particle such as a VLP, a bacteriophage or a virus and at least one attachment site connected to the core particle by at least one covalent bond. The (ii) antigen or antigenic determinant has at least one second attachment site which can associate with the first attachment site to form an ordered and repetitive antigen.  

To date, VLPs have been produced for more than 30 different viruses that infect humans and other animals. One of the most striking features of this group is that it is extremely diverse in terms of the structure of the individual viruses. (Noad, “Virus-like particles as immunogens” Trends in Microbiology, 11(9), 438-444, 2003). 

helpful websites:  NIH Vaccines    Immunisation   Immunisation Guidelines for Ireland 2008 Edition, Royal College of Physicians of Ireland (good summary of all of the vaccine schedules) GAVI: the vaccine alliance   International Society of Travel Medicine (find travel clinicls worldwide)  Yellow Fever Vaccination Clinics (if you are travelling to where yellow fever is endemic –CDC).  CDC Travel Advisory. 

Coalition for Epidemic Preparedness Innovations (CEPI)

 List of Vaccines approved by the FDA  Vaccine Schedule (birth-6 year old)  Vaccine Schedule 7-18

Companies: Bavarian Nordic. VaxArt (oral vaccines)

Vaccines are a solution or suspension of materials used to induce artificial active immunity by stimulating the immune system with an antigen that will prevent the disease. Vacines expose the body to a disabled form of a pathogen (or harmless pieces of it), but the immune system reacts as it would to a true assault, generating protective membory cells in the process. Antigens may be organisms, toxoids (modified toxin, rendered nontoxic), part of organisms (capsules) and nucleic acids. The ideal vaccine is one that is specific, safe, long lasting, no side effects, stable, easy to produce and easy to administer. The types of vaccines include toxoids which are immunogenic but not toxic, macromolecules from pathogens and recombinant vectors. For example, one can take genes which code for surface antigens for say hepatitis B and transfect yeast cells which will produce those antigens. Recombinant virus vacines are safe in that there is no problem with reversion. As a disadvantage, the immune repertoire is directed against only a small part of virus protein. 

Vaccination has virtually eradicated diseases such as polio, tetanus, tuberculosis, chicken pox, measles, hepatitis, etc. The approach using vaccinations has exploited the ability of the immune system to prevent infectious diseases. Vaccination with non-live materials such as proteins generally leads to an antibody response or CD4+ helper T cells response (Raychaudhuri and Morrow (1993), Immunology Today 14). On the other hand, vaccination with live materials such as live cells or infectious viruses generally leads to a CD8+ cytotoxic T lymphyoctye response. A CTL response is crucial for protection against cancers, infectious viruses and certain bacteria. This poses a practical problem in that the only way to acheive a CTL response is to use live agents which are themselves patholgenic. The problem has been dealth with by using an inactivated microorganism or one that is dead. Killed vaccines are easy to produce and safer. However, killed virus vaccines do not induce cellular immunity and have low levels of humoral immunity. They also require frequent boosting. A second approach is to use an  attenuated microorganism which is alive and immunogenic but not disease producing. Attenuation refers to the production of strains of pathogenic microorganisms which have essentially lost their disease producing ability. One way to accomplish this is to subject the microorganism to unusual growth conditions and/or frequent passage in cell culture. Mutants are then selected which have lost virulence but yet are capable of eliciting an immune reponse. Live-attenuated virus vacines replicate in vivo and induce cellular and humoral immunity. Disadvantages include risk of reversion to virulence. They are also expensive to transport in that they are heat/light sensitive.

Vaccines are often formulated to contain various adjuvants in addition to antigen. Adjuvants aid in attaining a more durable and higher level of immunity using smaller amounts of antigen or fewer doses than if the antigen were adminsitered alone. 

Recommended Vaccination Schedule

Vaccination and number of injections should be given at the following ages: (See National Immunisation Offce, February 2011 “”Frequently asked questions for Healthcare Professionals in Relation to Vaccine Scheduling for Children” ) see Royal C

2 months: (6 in 1 = Diptheheria, Tetanus acellular Pertussis (whooping cough), inactivted polio, Haemophilus Influenze B, Heapatis B) + Pneyococcal Conjugate Vacine (PCV)),

4 months (6 in 1 + Meningococcal C vaccine (Men C))

6 months: 6 in 1 + PCV + Men C . 3 injections.  Give 6 in 1 and Men C in the same limb (different sites and 2.5 cms apart) and give PCV in the other limb

2 months: Measals, Mumps Rubella (MMR) + PCV .  2 injections

13 months: Men C + Haemophilus Influenzae B (Hib) 

Passive Immunization

Injection of purified antibody or antibody-containing serum for rapid, temporary protection or treatment of an individual is called passive immunization. Newborns receive natural passive immunity from maternal immunoglobulin that has crossed the placenta or is present in the mother’s milk. Passive immnization with monoclonal antibdies has several advantages over active vaccination. For one, it allows treating people which poorly respond to vaccines, such as the elderly, young children or immune compromised individuals. In addition, it is the treatmnet of choice in situations where rapid protection is crucial, such as for post exposure treatment or prophylaxis for the acutely exposed.

Human serum globulin is prepared from pooled plasma and contains the normal repertoire of antibodies for an adult. It is preferable to animal immunoglobulin because there is less risk of a hypersensitivity reaction (serum sickness).

Types of Vaccines

Recombinant Protein Vaccines: 

RNA Vaccines: (injecting RNA into body)

–SARS:  no vaccine to this vrius

–Polio: several vaccines. The US uses inactivated vaccine (e.g., polio -injectable version) and other countires use an attentuated vaccine (e.g., measles mumps rubella).

–Influenza:  One is intramuscular and another nasal form.

DNA Vaccines:  a genetically engineered plasmid contianing a DNA sequence that encodes viral protein(s) introduced into body. 

–West Nile Virus Vaccine:  only veterinary use

Universal Vaccines:

One challenge to vaccine development is constructing vaccines that both incorproate and attack multiple virus strains.

Longhorn Vaccines and Diagnostics has patented a composite peptide approach that enables vaccines to string epitopes together into unique peptides. They find conserved epitopes in each target, put them in an optimal order and attach T cell epitopes to simulate the immune system further. (kaumaya PT “Peptide Vacines incorporating a “promiscuous” T-cell epitope bypass certain haplotype restricted immune reposnes and provide broad spectrum immunogenicity” J. Mol. Recognit. 6(2) 1993). 

 Vectors used to Get Vaccines into the Cell

Adenovirus Vector Vaccines: