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The Size of Viruses: Implications for Prophylactic Measures

Viruses are extremely small pathogens whose known species range in size from about 20 nm to several hundred nanometers (Figure 1). Yet they have the potential to affect our lives dramatically, as the SARS-CoV-2 epidemic has shown [1]. Here we describe how their size should be taken into account to develop and produce efficient prophylactic measures, from the fine-tuning of protective equipment and understanding how they interact with our immune system to the design and production of antiviral vaccines. We will show how our measurement technologies can be useful in performaning these challenging R&D and quality control tasks.

Figure 1: Human viruses range in size from 20 nm to several hundred nanometers. © ViralZone, SIB Swiss Institute of Bioinformatics, distributed under a CC BY 4.0 license.

Impact of the Size of Viruses on the Development of Protective Equipment

Strategies designed to limit contact with the pathogen, such as physical distancing or the use of protective equipment, are the first line of defense against viral outbreaks. 

When the route of entry of the virus is our airways, knowledge of how the virus is transmitted in the air is key to applying effective strategies. Viruses can only survive in an aqueous environment, so they are in fact always transmitted through the aqueous particles we naturally excrete, and not as single particles. 

Here, epidemiology teaches us that the minimum size of the aqueous particles in which the virus retains its infective potential makes a huge difference to the rate of transmission [2]. Droplets is the term used to describe respiratory particles larger than 5 µm. They are mostly produced when we talk, scream, sing or sneeze, do not project very far, and tend to fall to the ground quickly. Particles below 5 µm in diameter are termed aerosols because they have the ability to remain airborne for much longer periods than the droplets and can travel much longer distances. Thus, respiratory viruses that are able to retain infectious potential in aerosols generally have a much higher transmission rate than those that can only retain infectivity in droplets. 

In this regard, the SARS-CoV-2 pandemic has proved a textbook case. At the beginning of the pandemic, as virus transmission was thought to be through droplets alone, mitigating measures such as hand disinfection and surgical masks were regarded as sufficient to prevent the spread of the disease. As it became increasingly clear that transmission was also airborne, recommendations became more stringent, with countries such as Austria making the wearing of respirator masks (N95 or FFP2) compulsory in enclosed public spaces [3]

Respirator masks are tested for filtering efficacy using sodium chloride particulate aerosols and are qualified to filter ≥95 % of particles over 300 nm [4]. While they offer significant protection to the wearer, they do not constitute an absolute barrier against a virus contained in a nanometer-range aerosol. This is why the SARS-CoV-2 pandemic has also seen the rapid development of portable HEPA filters, which are qualified to retain ≥99.97 % of particles below 300 nm, for room air purification [5]

Water-borne viruses, which generally use our digestive system as a portal of entry, constitute a different challenge. Poliovirus, hepatitis A, and noroviruses are amongst the smallest known human viruses, averaging about 30 nm in diameter (Figure 1). As they do not use carrier particles, but are directly infectious, a water filtration system must ensure that particles in the size range of the virus itself are blocked. 

Here, progress is being made with the development of innovative nanofilters (e.g., nanofibrillated cellulose filters), which have added specific functionalization to increase virus retention. As viruses tend to be negatively charged, functionalization aimed at increasing the net charge of the filter membrane can increase their filtering capacity. In this regard, Anton Paar’s SurPASS 3 electrokinetic analyzer is used to characterize the zeta potential of antiviral water filters [6] [7]

Vaccine Development, Vaccine Quality Control: Why Particle Size Matters

Vaccination is widely acknowledged as the most-efficient prophylactic intervention against viruses. Here again, the size of the vaccine candidate has a significant impact on both its efficacy and its manufacturing process.

Figure 2: The size of a pathogen or a vaccine influences its immunogenicity. Adapted from [8]. CC BY 4.0 licensed.

In vaccine research and development, the last decade has seen a shift towards the use of nanoparticles as vaccine candidates. This has been driven by the increasing understanding of how our immune system picks up and processes pathogens. Particles in the nanometer size range are efficiently taken up by dendritic cells, a class of sentinel cells uniquely endowed with the ability to induce both antibody- and T cell-mediated immunity [9]. In addition, nanoparticles smaller than 200 nm have the capacity to passively cross the endothelial barrier and enter the lymph vessels, which in turns enables them to accumulate in lymph nodes [8]. As the bulk of the adaptive immune response takes place in our lymph nodes, this gives nanoparticulate vaccine candidates in the 10-200 nm range a significant efficacy boost (Figure 2). The recently developed technology of mRNA-liposome-based vaccines, which utilizes lipid nanoparticles of about 100 nm as carriers, has provided further evidence of this. In such a case, the particle size of the lipid nanoparticle carrier has a significant impact on the immunogenicity of the vaccine candidate [10]

Dynamic light scattering (DLS) is a fast and non-invasive measurement method that elucidates the size distribution of particles in the lower nanometer to lower micrometer size range. This makes DLS a method of choice for the development of antiviral vaccines. Anton Paar’s Litesizer 500 instrument has been used for the development of vaccine delivery systems as diverse as lipid nanoparticles, virus-like particles, and mineral-based or polymer-based nanoparticles. Litesizer 500 can also perform electrophoretic light scattering (ELS), which measures the particle charge – or zeta potential – of nanoparticles. This provides information on the colloidal stability of the nanoparticles, a key parameter in particular for lipid nanoparticle-based delivery systems [11]

Nowadays, DLS/ELS instruments are also a common sight in vaccine quality control laboratories. Not only can they ensure that the manufacturing steps have generated a product in the correct size range, they can also quickly and efficiently provide information about the presence of contaminants, aggregation behavior, and colloidal stability. 

Figure 3: Particle size distributions of different antiviral vaccines subjected to simulated cold-chain disruptions: heat treatment (50 °C, 18 hours) or freeze-thawing (1 cycle, -18 °C). Measured by DLS on a Litesizer 500 instrument. Curves represent the mean values from at least five measurement repetitions. CC BY 4.0 licensed.

Figure 3 shows DLS results obtained on three different antiviral vaccines subjected to simulated cold-chain disruptions. Samples of a tick-borne encephalitis (TBE) vaccine, consisting of inactivated virions adsorbed on alum microparticles, of a cell-based influenza vaccine, and of a SARS-CoV-2 mRNA/lipid nanoparticle vaccine were subjected to either an 18-hour incubation at 50 °C or a single freeze-thaw cycle.

Results show that the TBE vaccine significantly aggregates in response to freeze-thaw and even more markedly after heat-treatment. In contrast, the SARS-CoV-2 vaccine aggregates slightly after heat-treatment and more prominently after a single freeze-thaw cycle. The influenza vaccine shows the most complex aggregation behavior. The untreated sample displays a bimodal particle size distribution, with a minor peak at ca. 30 nm diameter likely representing fractionated virions, and a major peak around 250 nm showing viral aggregates. While a single freeze-thaw cycle leads to a significant decrease of the 30 nm peak (split virions), heat-treatment has the opposite effect and instead increases the proportion of split virions in the distribution – which suggests de-aggregation or fragmentation. Crucially, even differences that appear subtle in the particle size distribution graphs prove statistically significant due to the excellent repeatability of the technique. 

Beyond the product itself, vaccine quality control should also include an evaluation of the properties of the storage container. The protein and lipid components of the vaccine are susceptible to adsorption on the vial or syringe walls, which can potentially lead to material loss or even product aggregation/degradation. Hence, characterization of the container’s inner surface zeta potential by the SurPASS 3 electrokinetic analyzer can help minimize reactogenicity. Of note, a measuring cell specific for standard syringes was developed for the SurPASS 3 to facilitate such measurements. 

References

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[8] M. F. Bachmann and G. T. Jennings, "Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns," Nature Reviews Immunology, vol. 10, pp. 787-796, 2010. 

[9] J. Jia, Y. Zhang, Y. Xin, C. Jiang, B. Yan and S. Zhai , "Interactions Between Nanoparticles and Dendritic Cells: From the Perspective of Cancer Immunotherapy," Frontiers in Oncology, vol. 8, p. 404, 2018. 

[10] K. J. Hassett , J. Higgins, A. Woods, B. Levy, Y. Xia, C. J. Hsiao, E. Acosta, Ö. Almarsson, M. J. Moore and L. A. Brito, "Impact of lipid nanoparticle size on mRNA vaccine immunogenicity," Journal of Controlled Release, vol. 335, pp. 237-246, 2021. 

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