World-wide administration of intramuscular (i.m.) mRNA-based vaccines, mRNA-1273 and BNT162b2 for SARS-CoV-2, have proven to be highly effective against SARS-CoV-2 infection [1,2] by eliciting robust T cell responses and strong antibody responses against the SARS-CoV-2 Spike protein. The anti-Spike antibody, in particular the receptor-binding domain (RBD)-specific antibody, is thought to protect against infection with the virus by binding to the RBD domain and neutralizing the binding of variants on the surface of the virus to host cells.

Nucleotide modifications, which block surveillance of the innate immune system against viruses, and lipid nanoparticles (LNP) development, which stabilize mRNA and prevent its breakdown by in vivo RNA nucleases, are two of the most important developments in the mRNA therapeutics field. Kariko and colleagues demonstrated that chemically modified nucleosides such as pseudouridine (Ψ), thiouridine (s2U), and 5-methylcytidine (m5C) resulted in a significant reduction in the immunogenicity of mRNA and its increased protein translation [3]. However, an even bigger advance was the development of delivery systems that not only helped to reduce RNA degradation by extracellular nucleases, but also efficiently delivered mRNA to the cytoplasm of target cells where it could result in protein translation. An early problem with particulate systems such as LNP that were taken up by endocytosis was the breakdown of RNA in endosomes. This was solved by the synthesis of ionizable lipids, which, following their endocytosis, interacted with the anionic lipids of the endosomal membranes, promoting the intracellular release of the entrapped mRNA into the cytoplasm where protein translation occurred [4]. Vaccines composed of chemically modified SARS-CoV-2 Spike mRNA, and LNP delivery systems designed to released endocytosed mRNA to the cytoplasm where it was translated [5], have led to the efficient translation of mRNA, and immune responses.

Most of the clinically approved LNP consist of four lipid components, namely ionizable lipid, phospholipid, cholesterol, and a small amount (≤ 1.5 %) of polyethylene glycol (PEG)-lipid. The PEG-lipid component is used to modify the surface of the LNP by adding a protective hydrophilic layer that inhibits aggregation of LNP, improving their storage stability [6]. However, higher amounts (≥ 3–5 mol%) of PEGylation can reduce the cellular interactions of LNP with cells, interfering with their endocytosis, decreasing the activity of mRNA and small interfering RNA (siRNA) delivered by LNP [7,8]. PEG-lipids used in LNP for mRNA vaccines are modified to include shorter (14-carbon), dialkyl chains that rapidly dissociate from the LNP in vivo, allowing them to bind to cells, be endocytosed and be translated [9].

PEG has long been thought of as a poorly immunogenic compound in vivo, but numerous reports have demonstrated the presence of anti-PEG antibodies in the blood of individuals who had never received PEGylated drug formulations such as G-LASTA®, ONCASPAR®, ADAGEN®, KRYSTEXXA®, MIRCERA®, DOXIL®, ONYVIDE®, ONPATTRO® and mRNA vaccines, all of which contain PEG covalently attached to proteins or structural lipids [10,11]. In cosmetics, free PEG or PEG derivatives are widely used as emulsifiers and skin penetration enhancers. In particular, lotions, shampoos, make-ups, cleansing oils, sunscreens, toothpaste, body soap, hair spray, and other products that are extensively used, contain PEG and PEG derivatives. We recently showed that the topical application of a cosmetic product containing PEG derivatives induced anti-PEG IgM in mice [12]. Skin injuries or pathologies including sunburns, lacerations, abrasions, surgical wounds, acne, or shaving may increase skin penetration of free PEG and/or PEG derivatives into the systemic circulation. Thus, casual exposure to PEG compounds may be an important contributor to the source of anti-PEG antibodies in healthy individuals.

If circulating levels of pre-existing anti-PEG IgM antibodies are present when PEGylated therapeutics are administered systemically, they may bind to the therapeutics in the circulation and alter the pharmacokinetic properties, resulting in reduced therapeutic efficacies [13,14]. Moreover, in the clinic, anti-PEG antibodies have been reported to be related to rare instances of severe hypersensitivity reactions following the systemic administration of PEGylated therapeutics [15,16]. However, mRNA vaccines, administered i.m. into the deltoid muscle, are mainly taken up by muscle cells, stimulating antigen production at the injection site and the draining axillary, apical, and supraclavicular lymph nodes, resulting in the subsequent cellular and humoral immunogenic responses [17]. Currently, we don't know enough about the impact of pre-existing anti-PEG IgM on therapeutic outcomes when PEGylated therapeutics such as mRNA vaccines are administered i.m.

Skeletal muscle is highly vascularized [18], receiving ∼25 % of the cardiac output at rest [19]. In the event of skeletal muscle injury, an inflammatory response may ensue, characterized by the rapid invasion of the affected tissue by phagocytes, lymphocytes, immunoglobulin, and other serum proteins from the bloodstream. Hence, circulating anti-PEG antibodies may extravasate into muscles when i.m. injections of PEGylated mRNA vaccines are given. The anti-PEG antibodies could then, in theory, make immune complexes with the PEGylated mRNA vaccines, inhibiting them from interacting with intramuscular cells. This could hypothetically attenuate production of specific antibodies by suppressing antigen protein translation. Following the global success of mRNA anti-viral vaccines, the development of i.m. vaccines for other applications, such as cancer and infectious diseases, is progressing rapidly [20,21], so the i.m. route of administration for LNP formulations is expected to become increasingly important. A more detailed understanding of how anti-PEG antibodies affect responses to mRNA vaccines is required.

In this study, we investigated the effect of pre-existing anti-PEG IgM on the pharmacokinetics and efficacy of i.m. administered mRNA-loaded LNP (mRNA-LNP). Our results showed almost no effects of pre-existing anti-PEG IgM on in vivo LNP distribution and mRNA translation at injection sites and no attenuation of specific antibody induction to SARS-CoV-2 Spike protein, used as a model antigen. On the other hand, pre-existing circulating anti-PEG IgM did appear to affect the biodistribution and translation of mRNA-LNP extravasated into the circulation following i.m. administration.